Artificial nucleic acids for RNA editing

Abstract

The present invention provides an artificial nucleic acid for site-specific editing of a target RNA, which has enhanced editing specificity and avoids undesired off-target editing. The artificial nucleic acid comprises a targeting sequence comprising a nucleic acid sequence that is complementary or at least partially complementary to a target sequence in the target RNA that includes one or more nucleotides to be edited, wherein the targeting sequence is flanked by a first recruitment moiety capable of recruiting a deaminase and a second recruitment moiety capable of recruiting a deaminase, and at least one of the first and second recruitment moieties comprises at least one recruitment sequence, preferably two recruitment sequences, that binds to a complementary region in the target RNA.

Description

Detailed description of the invention

[0001] The present invention relates to artificial nucleic acids for site-specific editing of target RNA. In particular, the present invention provides artificial nucleic acids that can site-specifically edit endogenous transcripts by utilizing endogenous deaminases. Furthermore, the present invention provides artificial nucleic acids for site-specific editing of target RNA that provide enhanced editing specificity and avoid undesirable off-target editing. The present invention also includes a method for producing the above artificial nucleic acids, wherein at least some steps of the method are computer-assisted or computer-executed. The present invention also provides vectors encoding the above artificial nucleic acids, as well as cells, compositions, and kits containing the above artificial nucleic acids. Furthermore, the present invention provides the use of artificial nucleic acids, vectors, cells, compositions, or kits for site-specific editing of target RNA or for in vitro diagnostics. In addition, the artificial nucleic acids, vectors, cells, compositions, or kits described herein are provided for use as pharmaceuticals or for use in the diagnosis of diseases or disorders.

[0002] [Background technology] Conventional gene therapy typically manipulates genetic information at the DNA level, thus permanently altering the genome. Depending on the application, persistent genomic modification can be either beneficial or carry serious risks. In this respect, targeting RNA instead of DNA is an attractive alternative approach. When treating a target at the RNA level, changes in gene expression are usually reversible, tunable, and very often more efficient. On the one hand, the limited duration of effect also limits the risks associated with adverse side effects. Furthermore, if the effect can be fine-tuned, it becomes possible to continuously adjust the treatment in a time- and dose-dependent manner to control adverse effects. In addition, many manipulations of gene expression at the genomic level are impractical or ineffective, for example, if the gene deletion is lethal or easily compensated for by redundant treatment. Targeting signaling networks at the RNA level, for example, seems particularly attractive. Many signaling cues are essential or heavily overlapping, so knockout may not result in a clear phenotype, while knockdown may.

[0003] Therefore, there is growing interest in designing RNA targeting strategies. One such strategy is RNA editing. Adenosine (A)-inosine (I) RNA editing is a natural enzymatic mechanism for diversifying the transcriptome. Since inosine is biochemically interpreted as guanosine, editing from A to I formally introduces a mutation from A to G, which can result in recoding of amino acid codons, start and stop codons, splicing changes, and changes in miRNA activity. Targeting such enzymatic activity to specific sites in selected transcripts is a strategy called site-directed RNA editing, and it holds great promise for disease treatment and the study of protein and RNA function in general. RNA editing strategies based on modified deaminases have been developed (see, for example, Vogel, P., Schneider, MF, Wettengel, J., Stafforst, T. Improving Site-Directed RNA Editing In Vitro and in Cell Culture by Chemical Modification of the GuideRNA. Angew. Chem. Int. Ed. 53, 6267-6271 (2014)). However, in the therapeutic field, the use of broadly expressed endogenous deaminases that act on RNA seems most attractive. This would allow for the introduction of specific mutations into the transcriptome by administering oligonucleotide drugs alone, without requiring the ectopic expression of any (modified) protein. For example, Wettengel et al. (Wettengel, J., Reautschnig, J., Geisler, S., Kahle, PJ, Stafforst, T.: Harnessing human ADAR2 for RNA repair - Recoding a PINK1 mutation rescues mitophagy. Nucl. Acids Res. 45, 2797-2808 (2017)) have reported a system that uses cellular ADAR2 without requiring artificial proteins.Furthermore, oligonucleotide constructs for site-directed RNA editing are described in international patent applications WO2016 / 097212 and WO2017 / 010556. Additionally, German patent DE10 2015 012 522 B3 describes a guide RNA molecule for site-directed RNA editing.

[0004] Liang Qu et al. (Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nature Biotechnology, 37, 133-138 (2019)) constructed a long, unstructured gRNA that recruits human wild-type ADAR to some extent, but has significant problems with bystander off-target editing.

[0005] Cox et al. (Cox DBT, et al.: RNA editing with CRISPR-Cas13. Science 358, 1019-1027 (2017)) constructed ADAR recruit RNA by fusing the deaminase domain of the highly active E1008Q mutant ADAR1 to an RNA-guided RNA-targeting CRISP effector.

[0006] However, strategies known in the art have similar problems. On the one hand, it has been found difficult to efficiently recruit sufficient deaminase, particularly endogenous deaminase, to provide adequate RNA editing. On the other hand, efficient editing typically involves low specificity, such as off-target editing of numerous bystanders within the gRNA-mRNA double strand, as well as global off-target editing across the entire transcriptome. In addition, strategies known in the art have extremely limited sequence space because their gRNAs simply bind inversely complementary manner around the target adenosine within the target mRNA.

[0007] Merkle et al. (Merkle, T. Merz, S., Reautschnig, J., Blaha, A., Li, Q., Vogel, P., Wettengel, J., Li, J., Stafforst, T.: Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nature Biotechnology, 37, 1059-1069 (2019)) described the structure of chemically modified antisense oligonucleotides (guide RNAs) that recruit endogenous human ADARs and edit endogenous transcripts in a more specific manner. However, due to the chemical modification, the antisense oligonucleotides used in this approach cannot be synthesized by the organism itself and must be administered to the target.

[0008] Therefore, there is an urgent need for RNA editing strategies that enable high editing yield and high specificity without resulting in off-target editing. In particular, there is a need for compounds that are suitable for recruiting endogenous deaminases and can be expressed by themselves based on a vector encoding a guide nucleic acid, rather than being individually added, for example, by injection.

[0009] Therefore, an object of the present invention is to provide a compound that can recruit a deaminase, preferably an endogenous deaminase, such as adenosine deaminase, to an RNA target to be edited. A particular object of the present invention is to provide a compound suitable for editing RNA targets with high efficiency and high specificity, particularly with a reduced rate of off-target editing. Thus, an improved RNA editing approach is provided, which enables a high yield of RNA editing at a specific target site in the target RNA, preferably with or without a reduction in nonspecific editing at other transcriptome sites. Another particular object of the present invention is to provide a compound that can recruit a deaminase that may be endogenously expressed by the organism itself, preferably characterized by the advantages described above.

[0010] Solutions to the aforementioned objectives are achieved by embodiments described herein and defined by the claims.

[0011] [Detailed description of the invention] In a first embodiment, the present invention relates to a novel artificial nucleic acid for site-directed editing of target RNA. In particular, an artificial nucleic acid for site-directed editing of target RNA is provided herein, which comprises the following in the 5'-to-3' direction or the 3'-to-5' direction: a) A first recruitment portion capable of recruiting a deaminase, comprising at least one recruitment sequence that binds to a first region in the target RNA; b) A targeting sequence comprising a nucleic acid sequence that is complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited, and c) A second recruitment portion capable of recruiting deaminase, Here, the first region in the target RNA and the target sequence in the target RNA are separated by at least one nucleotide, the nucleotide is not bound by the at least one recruitment sequence, and is not complementary to the targeting sequence of the artificial nucleic acid.

[0012] The inventors have surprisingly found that artificial nucleic acids described herein, in particular artificial nucleic acids comprising two adjacent targeting sequences with two recruitment moieties, wherein at least one of the recruitment moieties comprises at least one recruitment sequence or cluster of recruitment sequences as defined herein, can recruit deaminase, particularly endogenous deaminase, to an RNA target and specifically edit nucleotides, preferably adenosine or cytidine nucleotides, at the target site in the RNA. Advantageously, the target RNA is edited with high efficiency by the artificial nucleic acids described herein, thus providing a high yield of edited target RNA while avoiding undesirable off-target editing. Thus, the artificial nucleic acids described herein enable site-directed RNA editing with both high efficiency and high specificity, opening up a large sequence space for guide RNA with more advantageous properties than state-of-the-art solutions.

[0013] The inventors have found that artificial nucleic acids are suitable for editing a wide variety of transcripts, such as endogenous mRNAs of housekeeping genes (e.g., NUP43, GUSB, PDE4D) and plasmid-encoding cDNA transcripts of disease-related genes (e.g., BMPR2 or COL3A1). Advantageously, the system according to the present invention has been demonstrated to be applicable to a wide variety of cells, ranging from immortalized cell lines and tumor cell lines to several primary human cells. For example, using the system of the present invention, the inventors successfully corrected disease-related hIDUA W402X amber mutations in primary fibroblasts derived from patients with Hurler syndrome. The use of multivalent recruitment clusters opens up a large sequence space for designing optimal guide RNAs for targeting any endogenous (m)RNA. Therefore, the inventors have found that recruitment sequences are effective even when bound to intronic or exonic sequences thousands of nucleotides apart on the target (m)RNA.

[0014] As used herein, the term “artificial nucleic acid (molecule)” typically refers to nucleic acids that do not exist in nature. In other words, an artificial nucleic acid molecule may be a non-natural nucleic acid molecule. Such an artificial nucleic acid molecule may be non-natural due to its individual sequences (sequences that do not exist in nature) and / or due to other modifications of nucleotides that do not exist in nature, such as structural modifications. As used herein, an artificial nucleic acid is preferably different from a naturally occurring nucleic acid by at least one nucleotide or at least one modification of a nucleotide. An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule, or a hybrid molecule containing both DNA and RNA portions. In a preferred embodiment, the artificial nucleic acid is an RNA molecule. In particular, the artificial nucleic acid as used herein may include unmodified or modified ribonucleotides and / or unmodified or modified deoxynucleotides, preferably unmodified ribonucleotides and / or deoxynucleotides. Furthermore, the term “artificial nucleic acid (molecule)” is not limited to a “single molecule” and may also refer to a collection of identical molecules. Thus, this expression may refer, for example, to multiple identical molecules contained in a sample.

[0015] In relation to the present invention, the term "RNA editing" refers to a reaction in which a nucleotide, preferably an adenosine nucleotide or a cytidine nucleotide, in a target RNA is converted to another nucleotide by a deamination reaction. The altered nucleotide preferably results in a change in codons, for example, the incorporation of another amino acid into a polypeptide translated from RNA or the generation or deletion of a stop codon, so that the change typically results in a different gene product. In particular, an adenosine nucleotide in the target RNA is converted to inosine by deamination, for example by an adenosine deaminase described herein. In another embodiment, a cytidine nucleotide in the target RNA is converted to a uridine nucleotide. As used herein, the term "target RNA" typically refers to RNA subjected to an editing reaction supported by the artificial nucleic acid described herein.

[0016] RNA editing achieved by the artificial nucleic acids described herein is further “site-specific,” meaning that a specific nucleotide at a target site in the target RNA is edited, preferably without editing, or essentially editing, other nucleotides. Typically, the nucleotide at the target site is targeted by a targeting sequence of the artificial nucleic acid described herein, where the targeting sequence can preferably form a specific base pair with the target sequence under physiological conditions. Thus, in relation to the present invention, the expression “target sequence” is used with respect to a nucleic acid sequence that is typically (at least partially) complementary to the targeting sequence of the artificial nucleic acid. The target sequence comprises a target site, where the target site is typically a nucleotide to be edited, preferably an adenosine nucleotide or a cytidine nucleotide. In some embodiments, the target site may comprise two or more nucleotides to be edited, where these nucleotides are preferably separated from each other by only at least one, preferably two other nucleotides. As used herein, the terms “complementary” or “partially complementary” refer to nucleic acid sequences that, preferably under physiological conditions, are capable of forming a specific intermolecular base pair, preferably a Watson-Crick base pair, for their complementary nucleotides. As used herein, the term “complementary” may also refer to a reverse complementary sequence. The artificial nucleic acids described herein may also be referred to herein as “antisense oligonucleotides” or “ASOs,” since the artificial nucleic acids typically include a nucleic acid sequence in a targeted sequence that represents antisense of a nucleic acid sequence in a target RNA. With respect to the present invention, the term “guide RNA” may be used to refer to an artificial nucleic acid that preferably guides deaminase function to a target site.

[0017] Artificial nucleic acids for site-directed editing of target RNA are provided herein, and such artificial nucleic acids include the following in the 5'-to-3' direction or the 3'-to-5' direction: a) A first recruitment portion capable of recruiting a deaminase, comprising at least one recruitment sequence that binds to a first region in the target RNA; b) A targeting sequence comprising a nucleic acid sequence that is complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited, and c) A nucleic acid sequence that can bind to deaminase.

[0018] In this regard, the second recruitment portion capable of recruiting deaminase preferably includes a nucleic acid sequence capable of binding deaminase, preferably a nucleic acid sequence as defined herein, such as an R / G motif.

[0019] (Recruitment section) The artificial nucleic acid of the present invention comprises at least two recruiting moieties adjacent to the targeting sequence. With respect to the present invention, the term “recruiting moiety” refers to a portion of the artificial nucleic acid described herein that recruits deaminase and is typically covalently bound to the targeting sequence. Thus, such a “recruiting moiety” recruits deaminase to a target site in the target RNA, where the target RNA (and target site) is preferably recognized and bound in a sequence-specific manner by the targeting sequence and at least one recruiting moiety.

[0020] The artificial nucleic acid of the present invention comprises at least two recruitment moieties, a first recruitment moiety and a second recruitment moiety, wherein at least one of the first and second recruitment moieties is located at 3' of the targeting sequence and at least one of the first and second recruitment moieties is located at 5' of the targeting sequence.

[0021] It should be noted that the terms "first recruitment portion" and "second recruitment portion" are used to distinguish between the recruitment portions of the artificial nucleic acid located 3' and 5' of the targeting sequence, without implying any limitation on the properties of the first and second recruitment portions. In other words, the features described for the "first" recruitment portion can also be applied to the "second" recruitment portion, and vice versa.

[0022] (First recruitment portion) In the artificial nucleic acid of the present invention, the first recruitment portion comprises at least one recruitment sequence that binds to a first region in a target RNA. In a preferred embodiment, the at least one recruitment sequence of the artificial nucleic acid comprises a nucleic acid sequence that is complementary or at least partially complementary to the first region in the target RNA. Accordingly, the recruitment sequence of the first recruitment portion, together with the targeting sequence, directs a deaminase to a target site in the target RNA in a sequence-specific manner.

[0023] In a further preferred embodiment of the present invention, the first recruitment portion comprises a cluster of recruitment sequences comprising at least two recruitment sequences linked via a nucleotide linker. Preferably, the cluster comprises at least 3 recruitment sequences, for example 3 to 20 recruitment sequences, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 recruitment sequences, more preferably 3 to 10 recruitment sequences.

[0024] A recruitment sequence, more preferably a cluster of recruitment sequences, increases the binding affinity of the artificial nucleic acid molecule for the target RNA, compared to a guide nucleic acid that is complementary to the target RNA only in the target region comprising the nucleotide to be edited. Furthermore, the provision of multiple recruitment sequences that are complementary or at least partially complementary to multiple regions in the target RNA may also increase the probability of encounter between the artificial nucleic acid and the target RNA.

[0025] The two recruitment sequences contained in a cluster of recruitment sequences are either arranged immediately adjacent to each other or linked via a nucleotide linker. In other words, any nucleotide linker can preferably separate two recruitment sequences in a cluster of recruitment sequences. The nucleotide linker linking the recruitment sequences preferably does not bind to a region in the target RNA that is bound by or complementary to the (at least two) recruitment sequences of the artificial nucleic acid, and comprises at least one nucleotide that is not complementary to the region in the target RNA. The nucleotide linker linking the (at least two) recruitment sequences of the first recruitment moiety of the artificial nucleic acid may comprise any type of nucleotide, for example adenosine, guanosine, cytidine, uridine or thymidine nucleotides, and preferably comprises one or more adenosine nucleotides. The nucleotide linker linking the recruitment sequences may for example comprise 1 to 100 nucleotides, such as 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20 or 1 to 10 nucleotides, preferably 2 to 6 nucleotides, more preferably adenosine nucleotides.

[0026] When an artificial nucleic acid binds to a target RNA, the nucleotide linker that junctions the two recruitment sequences in the artificial nucleic acid "crosslinks" nucleotides of the target RNA that are not complementary to the recruitment sequences and therefore not bound by the recruitment sequences. However, it should be noted that the number of nucleotides located between two regions on the target RNA bound by two individual recruitment sequences generally does not correspond to the number of nucleotides in the nucleotide linker that junctions the recruitment sequences in the artificial nucleic acid. For example, the distance between two regions on the target RNA that function as binding sites to two adjacent recruitment sequences of the artificial nucleic acid can include hundreds or even thousands of nucleotides, while the two adjacent recruitment sequences are separated by a fairly short, preferably defined, nucleotide linker. In this particular case, during the process of binding of the recruitment sequences to complementary regions of the target RNA, the regions of the target RNA located between the two specific binding sites of the recruitment sequences may form a loop in the target RNA. The feasibility of using recruitment sequences over such a wide range of distances (large sequence space) highlights the flexibility of the present invention compared to the latest technology.

[0027] At least one recruitment sequence of the first recruitment moiety may contain any number of nucleotides that bind to a nucleotide in the first region of the target RNA and are preferably complementary to the nucleotide in the first region of the target RNA. Preferably, the (at least one) recruitment sequence contains at least 10, preferably at least 15, and more preferably at least 20 nucleotides. In a preferred embodiment of the present invention, the at least one recruitment sequence contains 10 to 200 nucleotides, more preferably 10 to 100 nucleotides, or 15 to 100 nucleotides, or 20 to 100 nucleotides. Preferably, at least one recruitment sequence of the first recruitment moiety exists as essentially single-stranded nucleic acid, particularly under physiological conditions.

[0028] As described above, in a preferred embodiment, the first recruitment portion of the artificial nucleic acid includes a cluster of recruitment sequences comprising at least two recruitment sequences. Thus, in a preferred embodiment, the first recruitment sequence includes a nucleic acid sequence that binds to a first region in the target RNA, preferably complementary or at least partially complementary to the first region in the target RNA, and the second recruitment sequence or further recruitment sequence includes a nucleic acid sequence that binds to a second or further region in the target RNA, preferably complementary or at least partially complementary to the second or further region in the target RNA. For example, in a particular embodiment, the first recruitment portion includes three recruitment sequences, the first recruitment sequence binding to a first region in the target RNA and preferably including a nucleic acid sequence complementary or at least partially complementary to the first region in the target RNA; the second recruitment sequence binding to a second region in the target RNA and preferably including a nucleic acid sequence complementary or at least partially complementary to the second region or further regions in the target RNA; and the third recruitment sequence binding to a third region in the target RNA and preferably including a nucleic acid sequence complementary or at least partially complementary to the third region in the target RNA.

[0029] Multiple recruitment sequences, forming a cluster of recruitment sequences in the first recruitment region of the artificial nucleic acid, thus, together with the targeting sequence, guide the deaminase to the target site in a sequence-specific manner, thereby increasing the binding affinity of the artificial nucleic acid to the target RNA. Due to the large sequence space available for each recruitment sequence, their selection is highly flexible, thus allowing for the optimization of various important properties of the guide RNA, including editing efficiency, specificity, bystander editing, as well as stability, immunogenicity, toxicity, and others.

[0030] Preferably, the first region of the target RNA bound by the first recruitment sequence of the first recruitment portion, and / or the second and / or further regions of the target RNA bound by the second and / or further recruitment sequences of the first recruitment portion, do not contain editable adenosine nucleotides. That is, the first and further regions of the target sequence preferably do not contain any adenosine nucleotides except for adenosine nucleotides in a specific codon context subject to reduced or absent RNA editing (e.g., adenosine nucleotides following guanosine nucleotides in a 5'-GA-3' context) or adenosine nucleotides at specific locations (e.g., within 5 nt from the 5' or 3' end of the recruitment sequence).

[0031] Therefore, the recruitment sequence of the first recruitment portion of the artificial nucleic acid is preferably uridine base depleted unless it is within 5 nt from either end (5' or 3') of the recruitment sequence or is in a 5'-NUS(S=C or G) context.

[0032] In certain embodiments of the present invention, the recruitment sequence of the first recruitment portion of the artificial nucleic acid preferably has a reduced uridine content. For example, the recruitment sequence of the first recruitment portion preferably contains 50% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less than 1% of the total number of nucleotides in the recruitment sequence, unless it is within 5nt of any end (5' or 3') of the recruitment sequence or is in a 5'-NUS (S=C or G) context. More preferably, the recruitment sequence of the first recruitment portion of the artificial nucleic acid contains no uridine bases unless it is within 5nt of any end (5' or 3') of the recruitment sequence or is in a 5'-NUS (S=C or G) context.

[0033] In this way, off-target editing events in the first and subsequent regions of the target RNA, as well as in the double-stranded region formed by the recruitment sequence, are avoided.

[0034] In a preferred embodiment, the artificial nucleic acid includes a nucleotide spacer between the first recruitment portion and the targeting sequence. The nucleotide spacer is preferably neither bound to nor complementary to i) the first region in the target RNA bound by the recruitment sequence adjacent to the targeting sequence of the artificial nucleic acid, and ii) the target sequence in the target RNA.

[0035] Any nucleotide spacer that may be located between the first recruitment moiety and the targeting sequence of the artificial nucleic acid may comprise any nucleotide, such as adenosine, guanosine, cytidine, uridine, or thymidine nucleotides, and preferably comprises (one or more) adenosine nucleotides. As described herein with respect to any nucleotide linker that links the recruitment sequences of the first recruitment moiety, any nucleotide spacer optionally located between the first recruitment moiety and the targeting sequence preferably comprises at least one nucleotide, such as 1 to 100 nucleotides, more preferably 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 nucleotides, and even more preferably 2 to 6 nucleotides, wherein the nucleotides are preferably adenosine nucleotides.

[0036] Therefore, the nucleotide spacer linking the targeting sequence and the first recruitment sequence in the artificial nucleic acid "cross-links" the nucleotides of the target RNA located between the target sequence (bound by the targeting sequence of the artificial nucleic acid) and the first region in the target RNA (bound by the first recruitment sequence of the artificial nucleic acid). Here again, it should be noted that the number of nucleotides located between the target sequence and the first region on the target RNA (bound by the targeting sequence and the first recruitment sequence, respectively) generally does not match the number of nucleotides in the nucleotide spacer. Rather, the distance between the region on the target RNA that functions as a binding site to the targeting sequence and the first recruitment sequence of the artificial nucleic acid can include, for example, hundreds or even thousands of nucleotides. In this particular case, during the process in which the targeting sequence and the first recruitment sequence bind to the complementary region of the target RNA, the nucleotides located between the specific binding sites of the targeting sequence and the first recruitment sequence will form a loop in the target RNA.

[0037] (Targeted sequence) The targeting sequences adjacent to the first and second recruitment portions of the artificial nucleic acid include nucleic acid sequences that are complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited. Preferably, the targeting sequences include nucleic acid sequences that are complementary or at least 60%, 70%, 80%, 90%, 95%, or 99% complementary to the nucleic acid sequence in the target RNA, where the complementary nucleic acid sequence in the target RNA includes the target site and preferably contains at least 10 nucleotides, for example, at least 12, at least 15, at least 18, at least 20, at least 22, at least 25, or at least 30 nucleotides. In preferred embodiments, the targeting sequences include 10 to 50, more preferably 16 to 40 nucleotides. Preferably, the targeting sequences of the artificial nucleic acid exist as essentially single-stranded nucleic acids, particularly under physiological conditions.

[0038] According to some embodiments, the targeting sequence of the artificial nucleic acid includes a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abase site at a position corresponding to the nucleotide to be edited in the target sequence. Preferably, the targeting sequence includes, preferably, the adenosine to be edited and a cytidine nucleotide that mismatches the adenosine to be edited at a position corresponding to the nucleotide to be edited in the target sequence. Preferably, the cytidine nucleotide that mismatches the adenosine to be edited is located at least 6 nucleotides away from either the 5' or 3' end of the targeting sequence. More preferably, the cytidine nucleotide that mismatches the adenosine to be edited is located outside the center of the targeting sequence. For example, in a targeting sequence consisting of 20 nucleotides, the cytidine nucleotide that mismatches the adenosine to be edited is located at position 8 relative to the 3' or 5' end of the targeting sequence, preferably the 3' end.

[0039] In some embodiments, the target site in the target RNA comprises two or more nucleotides to be edited, and these nucleotides are preferably separated from each other by at least one, preferably two, other nucleotides. In these embodiments, the targeting sequence may contain the above-mentioned nucleotides, preferably cytidine nucleotides or variants thereof, at each position corresponding to the nucleotides to be edited.

[0040] The individual nucleic acid sequences of the targeting sequences of an artificial nucleic acid used to edit a given target RNA typically depend on the sequence of the target site of the specific target RNA being edited.

[0041] (Second recruitment section) In addition to the first recruitment moiety and targeting sequence, the artificial nucleic acid of the present invention includes a second recruitment moiety capable of recruiting a deaminase located at 3' or 5' relative to the targeting sequence. Thus, the artificial nucleic acid may comprise a structure 5'-first recruitment moiety-targeting sequence-second recruitment moiety-3' or 5'-second recruitment moiety-targeting sequence-first recruitment moiety-3'.

[0042] In one embodiment of the artificial nucleic acid, the second recruitment portion may be a recruitment portion defined with respect to the first recruitment portion. That is, the second recruitment portion may include at least one recruitment sequence that binds to a specific region in the target RNA and is preferably complementary to or at least partially complementary thereto, and more preferably includes a cluster of at least two recruitment sequences linked via any nucleotide linker defined above with respect to the first recruitment portion. In this particular embodiment, the artificial nucleic acid may optionally include nucleotide spacers between the targeting sequence and the second recruitment portion, preferably nucleotide spacers defined with respect to nucleotide spacers that may be present between the targeting sequence and the first recruitment portion.

[0043] However, in a preferred embodiment, the second recruitment portion includes a nucleic acid sequence that can bind to a deaminase, preferably an adenosine deaminase, without binding to the target mRNA. That is, in a preferred embodiment of the artificial nucleic acid, the first and second recruitment portions differ in their manner of recruiting the deaminase.

[0044] In certain embodiments, the second recruiting portion of the artificial nucleic acid comprises or consists of at least one coupling agent capable of recruiting a deaminase, the deaminase comprising a portion that binds to the coupling agent. The coupling agent that recruits the deaminase is typically covalently bonded to the 5' or 3' end of the targeting sequence. Alternatively, the coupling agent may be bonded to an internal nucleotide of the targeting sequence (i.e., not the 5'- or 3'-terminal nucleotide) via, for example, linkage to a nucleotide variant or modified nucleotide (preferably one of those described herein, such as aminothymidine).

[0045] The coupling agent is selected from the group consisting of O6-benzylguanine, O2-benzylcytosine, chloroalkanes, 1xBG, 2xBG, 4xBG, and any variant thereof. In a particularly preferred embodiment, the coupling agent is a branched molecule such as 2xBG or 4xBG, each of which is preferably capable of recruiting a deaminase molecule and therefore preferably amplifying the editing reaction. Exemplary structures of preferred branched coupling agents are shown below:

[0046] [ka]

[0047] The coupling agent can preferably bind specifically to a portion of the deaminase. The portion of the deaminase is preferably a tag, which is linked to the deaminase described herein, preferably an adenosine deaminase or cytidine deaminase described herein. More preferably, the tag is selected from the group consisting of SNAP-tag, CLIP-tag, HaloTag, and any one of these fragments or variants.

[0048] With respect to the present invention, a "variant" of a nucleic acid sequence or amino acid sequence is preferably identical to the sequence from which the variant is derived by at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. The variant is preferably a functional variant.

[0049] As used herein, a “fragment” of a nucleic acid sequence or amino acid sequence consists of a sequence of nucleotides or amino acid residues that corresponds to a sequence of nucleotides or amino acid residues in the full-length sequence, and this corresponds to at least 5%, 10%, 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length sequence from which the fragment is derived. In the sense of the present invention, such a fragment is preferably a functional fragment.

[0050] Therefore, in these embodiments, the deaminase coupled by the coupling agent is preferably an artificial version of an endogenous deaminase, preferably an artificial version of the deaminase described herein. Preferably, the deaminase is selected from the group consisting of SNAP-ADAR1, SNAP-ADAR2, Apobec1-SNAP, SNAPf-ADAR1, SNAPf-ADAR2, Apobec1-SNAPf, Halo-ADAR1, Halo-ADAR2, Apobec1-Halo, Clip-ADAR1, Clip-ADAR2, Clipf-ADAR1, Clipf-ADAR2, Apobec1-Clip, and Apobec1-Clipf, or selected from any fragment or variant thereof, where the deaminase is preferably derived from a human, mouse, or rat. More preferably, the deaminase is selected from the group consisting of SNAP-ADAR1, SNAP-ADAR2, SNAPf-ADAR1, SNAPf-ADAR2, Halo-ADAR1, Halo-ADAR2, Clip-ADAR1, Clip-ADAR2, Clipf-ADAR1, and Clipf-ADAR2, or selected from any fragment or variant thereof, wherein the deaminase is derived from humans. According to another embodiment, the deaminase is selected from the group consisting of mApobec1-SNAP, mApobec1-SNAPf, mApobec1-Halo, mApobec1-Clip, and mApobec1-Clipf, or selected from any fragment or variant thereof, wherein the deaminase is derived from mice.

[0051] In a particularly preferred embodiment, the deaminase is a hyperactive variant of any of the deaminases described herein, preferably a hyperactive Q variant, more preferably a hyperactive Q variant of ADAR1 deaminase, ADAR2 deaminase (e.g., human ADAR1p150, E1008Q; human ADAR1p110, E713Q; human ADAR2, E488Q), or a tagged version thereof, most preferably one of those described herein, or a fragment or variant of any of these.

[0052] In another embodiment, the deaminase is a variant of any of the deaminases described herein, the variant being one that changes the nucleotide specificity of the deaminase from adenosine to another nucleotide, for example, an ADAR2 deaminase variant capable of C- to -U RNA editing (see Abudayyeh, OO, et al.: A cytosine deaminase for programmable single-base RNA editing. Science 365(6451): 382-386. (2019)).

[0053] Preferably, the tagged deaminases described herein (e.g., SNAP-, SNAPf-, Clip-, Clipf-, Halo-tagged deaminases or fragments or variants thereof) are overexpressed for RNA editing, for example, by transient transfection of cells with a vector encoding the tagged deaminase, or by stable expression in transformed cells, tissues, or living organisms.

[0054] In certain embodiments, the second recruitment portion of the artificial nucleic acid comprises or consists of at least one RNA motif (e.g., MS2 loop, direct repeat of transactivating cr RNA, Box B motif, HIV transactivation response (TAR) hairpin), the RNA motif being a deaminase or other effector fusion protein developed for tethering approaches (similar MCP-ADARs (Azad, MTA, et al.: Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy. Gene Ther 24(12): 779-786 (2017), and D. Katrekar et al.: In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat. Methods 16(3), 239-242 (2019)); or similar dCas-ADARs (Cox, DBT, et al., supra; Omar O. Abudayyeh, et al., supra) or a similar LambdaN-ADAR (Montiel-Gonzalez, MF, et al.: Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. Proc Natl Acad Sci USA 110(45): 18285-18290(2013)) or a similar TBP-ADAR (S. Rauch et al.: Programmable RNA-Guided RNA Effector Proteins Built from Human Parts. Cell 178, 122-134.e12 (2019)) can be recruited.

[0055] In a preferred embodiment of the present invention, the second recruitment portion is a deaminase (preferably adenosine or cytidine deaminase, more preferably adenosine deaminase). The artificial nucleic acid according to the present invention contains or consists of a nucleic acid sequence that can specifically bind to the double-stranded (ds) RNA binding domain. Advantageously, the recruitment portion containing or consisting of a nucleic acid sequence that can bind to deaminase binds to endogenous deaminase. As a result, the artificial nucleic acid according to the present invention promotes site-directed RNA editing using endogenous (or heterologously expressed) deaminase.

[0056] Artificial nucleic acids are suitable for site-directed editing of RNA by deaminase, where the deaminase is preferably adenosine deaminase or a fragment or variant thereof, preferably ADAR (adenosine deaminase acting on dsRNA) enzyme or a fragment or variant thereof, more preferably selected from the group consisting of ADAR1, ADAR2 and their fragments or variants, for example, a peptide or protein containing an adenosine deaminase domain. However, cytidine deaminase or a fragment or variant thereof, such as Apobec1 or a fragment or variant thereof, for example, a peptide or protein containing a cytidine deaminase domain, is also intended.

[0057] As used herein, the term “deaminase” refers to any peptide, protein, or protein domain capable of catalyzing the deamination of nucleotides or variants thereof in target RNA, particularly the deamination of adenosine or cytidine. Therefore, the term refers not only to full-length and wild-type deaminases such as ADAR1, ADAR2, or Apobec1, but also to fragments or variants of deaminases, preferably functional fragments or functional variants. In particular, the term also refers to variants and variants of deaminases, such as variants of ADAR1, ADAR2, or Apobec1, preferably as described herein. Furthermore, as used herein, the term “deaminase” also includes any deaminase fusion protein (e.g., based on Cas9 and Cas13, MS2 coat protein, or Lambda-N-peptide, TAR-binding protein). In relation to the present invention, the term “deaminase” also refers to tagged variants of deaminases, such as deaminases selected from the group consisting of SNAP-ADAR1, SNAP-ADAR2, Apobec1-SNAP, SNAPf-ADAR1, SNAPf-ADAR2, Apobec1-SNAPf, Halo-ADAR1, Halo-ADAR2, Apobec1-Halo, Clip-ADAR1, Clip-ADAR2, Clipf-ADAR1, Clipf-ADAR2, Apobec1-Clip, and Apobec1-Clipf, or selected from any fragment or variant thereof, wherein the deaminase is preferably derived from humans, mice, or rats.

[0058] In preferred embodiments, the deaminase is an adenosine deaminase (such as ADAR1, preferably ADAR1p150 or ADAR1p110, or ADAR2), preferably a eukaryotic adenosine deaminase, more preferably a vertebrate adenosine deaminase, even more preferably a mammalian adenosine deaminase, most preferably a human adenosine deaminase such as hADAR1 or hADAR2, or a fragment or variant thereof.

[0059] According to another embodiment, the deaminase is a cytidine deaminase (such as Apobec1, preferably human Apobec1, murine Apobec1 (mApobec1), or rat Apobec1 (rApobec1)), for example, a vertebrate cytidine deaminase, preferably a mammalian cytidine deaminase, more preferably a eukaryotic cytidine deaminase such as murine or human cytidine deaminase, or a fragment or variant thereof. The deaminase may be a tagged cytidine deaminase, or a fragment or variant thereof. The deaminase may be selected from the group consisting of mApobec1-SNAP, mApobec1-SNAPf, mApobec1-Halo, mApobec1-Clip, and mApobec1-Clipf, or a fragment or variant thereof, where the deaminase is derived from a human, mouse, or rat.

[0060] In alternative embodiments, the deaminase is an overactive variant of any of the deaminases described herein (e.g., overactive Q variants of ADAR1 deaminase, ADAR2 deaminase (e.g., human ADAR1p150, E1008Q; human ADAR1p110, E713Q; human ADAR2, E488Q)) or a tagged version thereof, or a fragment or variant of any of these.

[0061] Preferably, the second recruitment portion includes or consists of a nucleic acid sequence that can specifically bind to the double-stranded (ds)RNA binding domain of the deaminase, wherein the nucleic acid sequence is preferably covalently bound to either the 5' or 3' end of the targeting sequence, more preferably to the 5' end of the targeting sequence.

[0062] In some embodiments, the recruiting portion includes or consists of a nucleic acid sequence capable of intramolecular base pairing. The recruiting portion preferably includes or consists of a nucleic acid sequence capable of forming a stem-loop structure. In certain embodiments, the stem-loop structure includes or consists of a double helix stem containing at least two mismatches. In preferred embodiments, the stem-loop structure includes a loop consisting of 3 to 8 nucleotides, preferably 4 to 6, and more preferably 5 nucleotides. The loop preferably includes or consists of the nucleic acid sequence GCUAA or GCUCA.

[0063] In a preferred embodiment, the second recruitment portion of the artificial nucleic acid contains the nucleotide sequence 5'-GGUGU CGAGA AGAGG AGAAC AAUAU GCUAA AUGUU GUUCU CGUCU CCUCG ACACC-3'. This nucleotide sequence has been found to be particularly effective in binding to ADAR1, especially to ADAR1p110.

[0064] In other embodiments, the second recruitment portion of the artificial nucleic acid comprises the nucleotide sequence 5'-GUG GAA UAG UAU AAC AAU AUG CUA AAU GUU GUU AUA GUA UCC CAC-3', and this nucleotide sequence in particular has been shown to be effective in binding to ADAR2.

[0065] The above sequence is applied from a well-known ADAR2 target site in glutamate receptor 2 mRNA and is therefore also known as the R / G motif.

[0066] Therefore, the second recruitment portion may be a recruitment portion defined with respect to the first recruitment portion, i.e., a recruitment portion comprising at least one recruitment sequence, preferably binding to a first region and preferably further regions in the target RNA, and preferably comprising a cluster of recruitment sequences that are complementary or at least partially complementary to said region, but preferably at least one of the first and second recruitment portions comprises a nucleic acid sequence that can bind to a deaminase, preferably a deaminase as described herein.

[0067] Therefore, the artificial nucleic acid of the present invention is preferably a single-stranded (ss) nucleic acid molecule. In a preferred embodiment, the artificial nucleic acid is a single-stranded nucleic acid containing a double-stranded (ds) region under physiological conditions. Preferably, the artificial nucleic acid is a single-stranded nucleic acid containing a double-stranded region within the recruitment region and is not intended to bind to a target mRNA that can bind to deaminase.

[0068] Therefore, in a preferred embodiment, the artificial nucleic acid of the present invention comprises the following in the 5'-to-3' direction or the 3'-to-5' direction: a) A first recruitment portion comprising at least one recruitment sequence that binds to a first region in the target RNA, preferably comprising a cluster of recruitment sequences that bind to the first region and further regions of the target RNA; b) A targeting sequence comprising a nucleic acid sequence that is complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited, and c) A second recruitment portion comprising a nucleic acid sequence capable of binding to a deaminase, preferably the nucleic acid sequence defined above, such as an R / G motif.

[0069] In a more preferred embodiment, the artificial nucleic acid of the present invention comprises, in the 3' to 5' direction, a) a first recruitment moiety, b) a targeting sequence, and c) a second recruitment moiety as defined above.

[0070] The second recruitment portion, a nucleic acid sequence capable of binding to a deaminase, preferably the nucleic acid sequence defined above, for example, including an R / G motif, may also include at least one recruitment sequence that binds to further regions in the target RNA, preferably complementary or at least partially complementary to those further regions, or clusters of recruitment sequences that each bind to further regions in the target RNA, preferably complementary or at least partially complementary to those regions.

[0071] In some embodiments, the artificial nucleic acids of the present invention include the following in the 5'-to-3' direction or the 3'-to-5' direction: a) A first recruitment portion comprising at least one recruitment sequence that binds to a first region of the target RNA, preferably complementary or at least partially complementary to the first region of the target RNA, or comprising a cluster of recruitment sequences that bind to the first region and further regions of the target RNA, preferably complementary or at least partially complementary to the first region and further regions of the target RNA; b) A targeting sequence comprising a nucleic acid sequence that is complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited, and c) A second recruitment moiety comprising at least one recruitment sequence that binds to a further region in the target RNA, preferably complementary or at least partially complementary to the further region in the target RNA, and a nucleic acid sequence capable of binding to a deaminase, preferably the nucleic acid sequence defined above, for example, comprising an R / G motif.

[0072] For example, the first recruitment portion may include one recruitment sequence that is complementary or at least partially complementary to a first region in the target RNA, while the second recruitment portion includes two recruitment sequences that are complementary or at least partially complementary to second and third regions in the target RNA, and a nucleic acid sequence that can bind to deaminase. In another embodiment, the first recruitment portion may include two recruitment sequences that are complementary or at least partially complementary to first and second regions in the target RNA, while the second recruitment portion includes a third recruitment sequence that is complementary or at least partially complementary to a third region in the target RNA, and a nucleic acid sequence that can bind to deaminase. Preferably, the nucleic acid sequence that can bind to deaminase, such as an R / G motif, is located at the 5' end of the artificial nucleic acid.

[0073] To avoid off-target editing events in the double-stranded region formed by further regions of the target RNA and the recruitment sequence, the recruitment sequence of the second recruitment portion is uridine base depleted unless it is within 5 nt of either end (5' or 3') of the recruitment sequence or is in a 5'-NUS (S=C or G) context.

[0074] Therefore, the recruitment sequence of the second recruitment portion preferably has a reduced uridine content. For example, the recruitment sequence of the second recruitment portion contains 50% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less than 1% of the total number of nucleotides in the recruitment sequence, unless there are uridine nucleotides within 5nt of any end (5' or 3') of the recruitment sequence or in any of the 5'-NUS(S=C or G) contexts. More preferably, the recruitment sequence of the second recruitment portion of the artificial nucleic acid contains no uridine bases unless they are within 5nt of any end (5' or 3') of the recruitment sequence or in the 5'-NUS(S=C or G) context.

[0075] In the target RNA, the first region (bound by at least one recruitment sequence of the first recruitment region) and the target sequence (complementary to the targeting sequence) are separated by at least one nucleotide that is not bound by at least one recruitment sequence and is not complementary to the targeting sequence of the artificial nucleic acid. That is, the first region and the target sequence in the target RNA preferably do not merge with each other. The first region and the target sequence in the target RNA may preferably be separated by at least one nucleotide, preferably 2 to 10,000 nucleotides, for example, 2 to 5,000 nucleotides, 2 to 1,000 nucleotides, 2 to 500 nucleotides, 2 to 400 nucleotides, 2 to 300 nucleotides, 2 to 200 nucleotides, or 2 to 100 nucleotides. More preferably, the first region and target sequence in the target RNA are separated by 2 to 50 nucleotides, for example, 2 to 40 nucleotides, 2 to 30 nucleotides, 2 to 20 nucleotides, or 2 to 10 nucleotides.

[0076] Similarly, in the target RNA, the first region (bound by the first recruitment sequence of the first recruitment portion) and the second or further region (bound by the second or further recruitment sequence of the first recruitment portion) are preferably separated by at least one nucleotide, preferably 2 to 10,000 nucleotides, for example, 2 to 5,000 nucleotides, 2 to 1,000 nucleotides, 2 to 500 nucleotides, 2 to 400 nucleotides, 2 to 300 nucleotides, 2 to 200 nucleotides, or 2 to 100 nucleotides. More preferably, the first region and the second or further region in the target RNA are separated by 2 to 50 nucleotides, for example, 2 to 40 nucleotides, 2 to 30 nucleotides, 2 to 20 nucleotides, or 2 to 10 nucleotides.

[0077] Therefore, the artificial nucleic acid of the present invention, which binds to the target RNA via at least one recruitment sequence and targeting sequence, is obtained over a region of 1,000, 10,000, or even 100,000 nucleotides on the target RNA, thereby recruiting deaminase to a specific target site on the target RNA. It should be noted that if the target sequence of the target RNA bound by the targeting sequence of the artificial nucleic acid is only a few hundred or thousands of nucleotides away from the first region bound by the first recruitment sequence of the artificial nucleic acid, for example, 100, 200, 300, 400, 500, 1,000, 5,000, 10,000, or 100,000 nucleotides, these nucleotides may form loops in the target RNA once the artificial nucleic acid is bound.

[0078] The artificial nucleic acid according to the present invention is not limited in length and may be, for example, an oligonucleotide. As used herein, the term “oligonucleotide” can refer to short nucleic acid molecules (e.g., hexamers or decamers) as well as longer oligonucleotides (e.g., nucleic acid molecules containing 100 or 200 nucleotides), where the oligonucleotide may include (unmodified or modified) ribonucleotides and / or (unmodified or modified) deoxynucleotides. According to a preferred embodiment, the artificial nucleic acid contains at least about 15, preferably at least about 20, more preferably at least about 25, even more preferably at least about 30, even more preferably at least about 35, and most preferably at least about 40 nucleotides. Alternatively, the length of the artificial nucleic acid is approximately 15 to 1000 nucleotides, for example, approximately 15 to 400 nucleotides, approximately 15 to 300 nucleotides, or approximately 15 to 200 nucleotides, preferably approximately 20 to 150 nucleotides, more preferably approximately 20 to 100 nucleotides, and most preferably approximately 20 to 80 nucleotides.

[0079] In certain embodiments of the present invention, the artificial nucleic acid may contain chemically modified nucleotides. As used herein, the term “chemical modification” refers to a chemical modification selected from skeletal modifications, sugar modifications, or base modifications, preferably including a debasement site. With respect to the present invention, “chemically modified nucleic acid” may refer to a nucleic acid containing at least one chemically modified nucleotide.

[0080] In certain embodiments of the present invention, the first recruiting moiety and / or targeting sequence and / or second recruiting moiety of the artificial nucleic acid may comprise at least one chemically modified nucleotide. In particular, the first recruiting moiety and / or targeting sequence and / or second recruiting moiety may comprise a plurality of chemically modified nucleotides that may result in a specific modification pattern as disclosed, for example, in WO / 2020 / 001793.

[0081] In general, the artificial nucleic acid molecules of the present invention may include natural (=naturally occurring) nucleotides as well as chemically modified nucleotides. As used herein, the term “nucleotide” generally includes (unmodified and modified) ribonucleotides and (unmodified and modified) deoxynucleotides. Therefore, the term “nucleotide” preferably refers to adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, deoxyinosine, 5-methoxyuridine, thymidine, uridine, deoxyuridine, cytidine, deoxycytidine, or their variants. Furthermore, when “nucleotide” is referred to herein, it is preferable that each nucleoside is also included.

[0082] In this regard, a “variant” of a nucleotide is typically a natural or artificial variant of a nucleotide. Therefore, a variant is a chemically derivatized nucleotide having a non-natural functional group that is added to or deleted from a natural nucleotide, or that substitutes a naturally occurring functional group of the nucleotide. Thus, in such a nucleotide variant, each part of the natural nucleotide (preferably a ribonucleotide or deoxynucleotide) may be modified; that is, the base portion, the sugar (ribose) portion, and / or the phosphate portion forming the backbone of the artificial nucleic acid may be modified, preferably by the modifications described herein. Therefore, the term “variant (such as a nucleotide, ribonucleotide, or deoxynucleotide)” also preferably includes a chemically modified nucleotide as described herein.

[0083] As used herein, chemically modified nucleotides are preferably variants of guanosine, uridine, adenosine, thymidine, and cytidine, including, but not limited to, natural or unnatural guanosine, uridine, adenosine, thymidine, or cytidine that have been chemically modified by, for example, acetylation, methylation, hydroxylation, etc., as well as 1-methyladenosine, 1-methylguanosine, 1-methylinosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurine riboside, 2-aminopurine riboside, 2'-araadenosine, 2'-aracytidine, 2'-arauridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'- Azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-fluorothymidine, 2-methyladenosine, 2-methylguanosine, 2-methylthio-N6-isopentenyladenosine, 2'-O-methyl-2-aminoadenosine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine, 2'-O-methyl-2'-deoxyguanosine, 2'-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O-methylinosine, 2'-O-methylpsoiduridine, 2-thiocytidine, 2-thiocytidine, 3-methylcytidine, 4-acetylcytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5,6-Dihydrouridine, 5-Aminoallylcytidine, 5-Aminoallyl-deoxyuridine, 5-Bromouridine, 5-Carboxymethylaminomethyl-2-thiouracil, 5-Carboxymethylaminomethyl-uracil, 5-Chloro-alacytosine, 5-Fluorouridine, 5-Iodouridine, 5-Methoxycarbonylmethyluridine, 5-Methoxyuridine, 5-Methyl-2-thiouridine, 6-Azacytidine, 6-Azauridine, 6-Chloro-7-deaza-guanosine, 6-Chloroprinriboside, 6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside, 7-Deaza-2'-deoxy-guanosine, 7-Deazaadenosine, 7-Methyl-gu Examples include anosine, 8-azaadenosine, 8-bromoadenosine, 8-bromoguanosine, 8-mercaptoguanosine, 8-oxoguanosine, benzimidazole-riboside, beta-D-mannosyl-queosin, dihydrouridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)adenosine, N6-isopentenyladenosine, N6-methyladenosine, N7-methylxanthosine, N-uracil-5-oxyacetate methyl ester, puromycin, queosin, uracil-5-oxyacetic acid, uracil-5-oxyacetate methyl ester, weybutoxosin, xanthosine, and xyloadenosine. Methods for preparing such variants are known to those skilled in the art and are described, for example, in U.S. Patents US4,373,071, US4,401,796, US4,415,732, US4,458,066, US4,500,707, US4,668,777, US4,973,679, US5,047,524, US5,132,418, US5,153,319, US5,262,530, or 5,700,642.

[0084] In some embodiments, the artificial nucleic acids described herein include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, and 4-thiouridine-5'-triphosphate. Acid, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine 5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azacitine-5'-triphosphate It comprises at least one chemically modified nucleotide selected from nic acid, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, or xanthosine-5'-triphosphate.

[0085] In some embodiments, the artificial nucleic acids described herein include pyridine-4-1-ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-psoiduridine, 2-thio-psoiduridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-psoiduridine, 5-propynyluridine, 1-propynyl-psoiduridine, 5-taurinomethyluridine, 1-taurinomethyl-psoiduridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5 It comprises at least one chemically modified nucleotide selected from -methyluridine, 1-methylpsoiduridine, 4-thio-1-methylpsoiduridine, 2-thio-1-methylpsoiduridine, 1-methyl-1-deaz-psoiduridine, 2-thio-1-methyl-1-deaz-psoiduridine, dihydrouridine, dihydropsoiduridine, 2-thio-dihydrouridine, 2-thio-dihydropsoiduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypsoiduridine, and 4-methoxy-2-thiopsoiduridine.

[0086] In some embodiments, the artificial nucleic acids described herein include 5-aza-cytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpsoidisocytidine, pyrrolo-cytidine, pyrrolo-psoidisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopsoidisocytidine, 4-thio-1-methylpsoidisocytidine, and 4 It comprises at least one chemically modified nucleotide selected from -thio-1-methyl-1-deazapseidoisocytidine, 1-methyl-1-deazapseidoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-psoidisocytidine, and 4-methoxy-1-methyl-psoidisocytidine.

[0087] In other embodiments, the artificial nucleic acids described herein include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyiso It comprises at least one chemically modified nucleotide selected from pentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0088] In other embodiments, the artificial nucleic acids described herein include at least one chemically modified nucleotide selected from inosine, 1-methyl-inosine, waiosine, waibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0089] In certain embodiments, the artificial nucleic acids described herein include 6-azacytidine, 2-thiocytidine, alpha-thiocytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methylpsoiduridine, 5,6-dihydrouridine, alpha-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, and alpha It contains at least one chemically modified nucleotide selected from alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, and 7-deaza-adenosine.

[0090] In certain embodiments, the artificial nucleic acid comprises at least one chemically modified nucleotide, wherein the 2' position is chemically modified. Preferably, the chemically modified nucleotide comprises a substituent at the 2' carbon atom, the substituent being selected from the group consisting of halogens, alkoxy groups, hydrogen, aryloxy groups, amino groups, and aminoalkoxy groups, and preferably selected from 2'-hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro. With respect to artificial nucleic acids, in particular, if the artificial nucleic acid is a molecule containing RNA or ribonucleotide, a 2'-deoxynucleotide (containing hydrogen as a substituent at the 2' carbon atom), such as deoxycytidine or its variants, may also be called a "chemically modified nucleotide."

[0091] Other chemical modifications of the 2' position of nucleotides described herein are locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and (S)-restricted ethyl cEt nucleotides. These skeletal modifications fix the sugar of the modified nucleotide to a preferred northern conformation. The presence of this type of modification in the targeting sequence of artificial nucleic acids is thought to enable stronger and faster binding of the targeting sequence to the target RNA.

[0092] According to some embodiments, the artificial nucleic acid comprises at least one chemically modified nucleotide, where the phosphate backbone incorporated into the artificial nucleic acid molecule is modified. The phosphate group of the backbone may be modified, for example, by substituting one or more oxygen atoms with other substituents. Furthermore, the modified nucleotide may include total substitution of the unmodified phosphate portion with a modified phosphate, as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, stereopure phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkylphosphonates, arylphosphonates, and phosphotryesters. The phosphate linker may also be modified by substituting the bound oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate), and carbon (bridged methylenephosphonate).

[0093] In a more preferred embodiment, the artificial nucleic acid includes a debasing site. As used herein, “debasing site” is a nucleotide lacking an organic base. In a preferred embodiment, the debasing nucleotide further includes the chemical modifications described herein at the 2' position of ribose. Preferably, the 2' position of the carbon atom of ribose is substituted with a substituent selected from the group consisting of halogens, alkoxy groups, hydrogen, aryloxy groups, amino groups, and aminoalkoxy groups, preferably a substituent selected from 2'-hydrogen (2'-deoxy), 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro. Thus, with respect to the present invention, “chemically modified nucleotide” may also be a debasing site.

[0094] In other embodiments, the artificial nucleic acid molecule may be modified by adding a so-called “5' cap structure.” The 5' cap is generally a component that “caps” the 5' end of mature mRNA, typically a component of a modified nucleotide. The 5' cap may typically be formed by a modified nucleotide, and in particular by a derivative of a guanine nucleotide. Preferably, the 5' cap is linked to the 5' end of the artificial nucleic acid via a 5'-5'-triphosphate bond. The 5' cap may be methylated, for example, m7GpppN, where N is the 5'-terminus nucleotide of the nucleic acid possessing the 5' cap, typically the 5' end of RNA. Further examples of 5' cap structures include glyceryl, inverted deoxydecate residue (partial), 4',5' methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbon cyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, alpha-nucleotide, modified base nucleotide, threopentofuranosyl nucleotide, acyclic 3',4'-seconucleotide, acyclic 3,4-dihydro The compounds include xybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted debase moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted debase moieties, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, or crosslinked or non-crosslinked methylphosphonate moieties.Particularly preferred modified 5' cap structures are CAP1 (methylation of the ribose of the nucleotide adjacent to m7G), CAP2 (methylation of the ribose of the second nucleotide downstream of m7G), CAP3 (methylation of the ribose of the third nucleotide downstream of m7G), CAP4 (methylation of the ribose of the fourth nucleotide downstream of m7G), ARCA (anti-reverse CAP analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0095] In some embodiments, the artificial nucleic acid includes a portion that enhances the intracellular uptake of the artificial nucleic acid. Preferably, the portion that enhances intracellular uptake is a triantennae-type N-acetylgalactosamine (GalNAc3) that is conjugated to the 3' or 5' end of the artificial nucleic acid.

[0096] In preferred embodiments of the present invention, the artificial nucleic acid comprises unmodified ribonucleotides and / or unmodified deoxynucleotides. More preferably, the artificial nucleic acid according to the present invention is RNA or an RNA analog. Most preferably, the artificial nucleic acid according to the present invention is endogenously expressible RNA.

[0097] In one embodiment, the present invention provides a method for generating a sequence of artificial nucleic acid for site-directed editing of a target RNA as defined above.

[0098] A method for generating an artificial nucleic acid sequence for site-specific editing of target RNA includes the following steps: i) Generate a sequence of a first recruitment portion, wherein the first recruitment portion comprises at least one recruitment sequence that binds to a first region in the target RNA; ii) Arbitrarily generate a sequence of nucleotide spacers containing at least one nucleotide; iii) Generate a targeted sequence that includes nucleic acid sequences that are complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited; iv) generate a sequence of a second recruiting portion capable of recruiting deaminase; and v) Assemble the sequence of the first recruitment portion, optionally a nucleotide spacer, a targeting sequence, and the sequence of the second recruitment portion in the 5' to 3' direction or the 3' to 5' direction.

[0099] In a preferred embodiment, steps i) and optionally iv) in a method for generating a sequence of artificial nucleic acids include generating a recruitment moiety comprising at least two recruitment sequences, preferably generating a cluster of recruitment sequences linked via a nucleotide linker.

[0100] The generated nucleic acid sequence can be expressed in vitro or in vivo, thereby synthesizing the artificial nucleic acid molecule of the present invention. The artificial nucleic acid synthesized based on the generated sequence is suitable for editing RNA targets with high efficiency and high specificity, particularly with a reduced rate of off-target editing, as described above.

[0101] The first recruitment portion and optionally the second recruitment portion of the artificial nucleic acid of the present invention include at least one recruitment sequence, preferably a cluster of recruitment sequences, that binds to a first region and optionally further regions of the target RNA. To avoid undesirable out-of-target events in the double-stranded region formed by the at least one recruitment sequence and the first and further regions of the target RNA, the target RNA bound by the at least one recruitment sequence preferably does not contain editable nucleotides, and more preferably does not contain editable adenosine nucleotides. Therefore, in order to generate the recruitment sequences of the first and optionally second recruitment portions of the artificial nucleic acid that bind to the first region and optionally further regions of the target RNA, the target RNA is preferably screened for regions that are suitable as binding sites to at least one recruitment sequence of the artificial nucleic acid and preferably do not contain editable adenosine nucleotides. Since the “manual” selection of the first and further regions can be very cumbersome, the screening step is preferably achieved by means of a computer program (“Recruitment Cluster Finder” (RCF)) described later.

[0102] Therefore, in the method for generating artificial nucleic acid sequences of the present invention, steps i) and optionally iv) are preferably achieved by a computer-based method comprising the step of screening a nucleic acid sequence for a target RNA to be edited, which is suitable as a binding site to at least one recruitment sequence and preferably does not contain an editable adenosine nucleotide.

[0103] When using a computer-operated method for generating the sequences of the first and optionally second recruitment portions of the artificial nucleic acid of the present invention, further specifications can be made, such as the length of a suitable nucleic acid sequence as a binding site to the recruitment sequence on the target RNA, and the distance between the binding sites to individual recruitment sequences on the target RNA, to generate beneficial or even more optimal sequences of the recruitment sequence.

[0104] In a more preferred embodiment of the method for generating artificial nucleic acid sequences of the present invention, all steps (i) to (v) are performed by a computer.

[0105] Preferably, in a computer-based method of the present invention, the sequence of the artificial nucleic acid of the present invention is generated based on a preset with respect to, for example, the following points. - Length of the nucleic acid sequence that functions as a binding site to the target sequence on the target RNA, - The distance between the binding site to the target sequence and the binding site to at least one recruitment sequence on the target RNA. - Length of the nucleotide linker, and length of any nucleotide spacer, - The length of a nucleic acid sequence suitable as a binding site to the recruitment sequence on the target RNA, and / or - The distance between binding sites to individual recruitment sequences on the target RNA.

[0106] As part of the preset used in the computer-based method of the present invention, the length of the nucleic acid sequence that functions as a binding site to a targeting sequence on the target RNA may be set to, for example, 5 to 100 nucleotides, for example, in the range of 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, or 5 to 40 nucleotides, preferably in the range of 10 to 50, more preferably in the range of 16 to 40 nucleotides.

[0107] Furthermore, the distance between the binding site to the targeting sequence (target sequence) and the binding site to at least one recruitment sequence (first region) in the target RNA is set to at least one nucleotide. Preferably, the distance between the target sequence and the first region in the target RNA is set to 2 to 100,000 nucleotides, for example, in the range of 2 to 100,000, 2 to 10,000, 2 to 5,000, 2 to 1,000, 2 to 500, 2 to 400, 2 to 300, 2 to 200, and 2 to 100 nucleotides, and more preferably in the range of 2 to 50 nucleotides.

[0108] Furthermore, the length of the nucleotide linker that connects the recruitment sequences of the recruitment cluster, and the length of the optional nucleotide spacer located between the first recruitment portion of the artificial nucleic acid and the targeting sequence, are set to at least one nucleotide. Preferably, the length of the nucleotide linker and the length of the optional nucleotide spacer are set to a range of 1 to 100 nucleotides, for example, 1 to 90 nucleotides, 1 to 80 nucleotides, 1 to 70 nucleotides, 1 to 60 nucleotides, 1 to 50 nucleotides, 1 to 40 nucleotides, 1 to 30 nucleotides, 1 to 20 nucleotides, or 1 to 10 nucleotides, and preferably to a range of 2 to 6 nucleotides.

[0109] As part of the presets used in the computer-based method, the length of a suitable nucleic acid sequence as a binding site to a recruitment sequence on the target RNA can be set to a range of at least 10 nucleotides (e.g., 10-500, 10-400, 10-300, or 10-200 nucleotides), or at least 20 nucleotides, preferably 20-100 nucleotides.

[0110] Furthermore, in the computer-based method for generating artificial nucleic acid sequences according to the present invention, the distance between individual binding sites to the recruitment sequence on the target RNA is set to at least one nucleotide, for example, in the range of 1 to 100,000 nucleotides (e.g., 1 to 100,000, 1 to 10,000, 1 to 5,000, 1 to 1,000, 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, or 1 to 100 nucleotides), preferably in the range of 2 to 500 nucleotides (e.g., 2 to 400, 2 to 300, 2 to 200, or 2 to 100 nucleotides), and more preferably in the range of 2 to 50 nucleotides.

[0111] Through the preset means described above, at least one nucleotide sequence is produced by a computer-operated method capable of synthesizing the artificial nucleic acid of the present invention. In a preferred embodiment, the computer-operated method for generating the sequence of the artificial nucleic acid of the present invention includes producing an alternative sequence of the artificial nucleic acid, in particular an alternative sequence of the first recruitment portion, which includes a cluster of recruitment sequences.

[0112] In a more preferred embodiment, the computer-based method for generating the artificial nucleic acid sequence of the present invention further includes the step of determining potential intramolecular base pairing events within the assembled artificial nucleic acid. In this way, among the constructed alternative sequences of potential artificial nucleic acids, the artificial nucleic acid exhibiting the smallest potential intramolecular base pairing can be detected and selected as an artificial nucleic acid molecule particularly suitable for site-directed editing of a specific target RNA.

[0113] For this purpose, a computer-based method further includes evaluating whether a selected recruitment sequence or cluster of selected recruitment sequences, preferably an artificial nucleic acid, could bind to another site on the target RNA or to another RNA molecule in the organism's transcriptome, resulting in undesirable mismatches and out-of-target events, and eliminating such recruitment sequences or clusters of such recruitment sequences.

[0114] In certain embodiments of the present invention, step iv) of the above method for generating an artificial nucleic acid sequence comprises the same steps as defined with respect to step i). That is, similar to the sequence of the first recruitment portion, the sequence of the second recruitment portion is generated as a sequence comprising at least one recruitment sequence, preferably a cluster of recruitment sequences that bind to a specific region on the target RNA. In other words, the artificial nucleic acid generated by the method of the present invention may comprise at least one recruitment sequence, preferably a cluster of recruitment sequences 3' and 5' of the target sequence.

[0115] However, as described above, in a preferred embodiment, the second recruitment portion of the artificial nucleic acid includes a nucleic acid sequence that can bind to deaminase. Therefore, in a preferred embodiment, a nucleic acid sequence that can bind to deaminase, preferably the nucleic acid sequence defined above, is preset as the second recruitment portion in the method for generating the sequence of the artificial nucleic acid of the present invention.

[0116] As described above, the first recruitment portion, optionally a nucleotide spacer, a targeting sequence, and the second recruitment portion can be assembled in the 5' to 3' direction or the 3' to 5' direction. Preferably, the sequences of the components of the artificial nucleic acid are assembled in the 3' to 5' direction.

[0117] In a preferred embodiment of the present invention, all of the above steps i) to v), as well as the further steps of determining potential intramolecular base pairing events and evaluating whether the artificial nucleic acid can bind to other sites on the target RNA or to other molecules in the transcriptome of a particular organism, are performed by computer, thereby producing an optimal sequence of the artificial nucleic acid for site-directed editing of a specific target RNA.

[0118] Therefore, a preferred method for generating an artificial nucleic acid sequence for site-specific editing of target RNA includes the following steps: i) A first recruitment segment sequence comprising at least one recruitment sequence, preferably comprising at least three, preferably three to ten, recruitment sequence clusters, Each of the aforementioned recruitment sequences comprises at least 10, preferably 15 to 100 nucleotides, and binds to a first region and further regions in the target RNA, and is linked via a nucleotide linker, more preferably an adenosine linker. Generate the array for the first recruitment portion; ii) Generate a sequence of nucleotide spacers containing at least one nucleotide, preferably two to six nucleotides, more preferably adenosine nucleotides; iii) Generate a targeted sequence comprising a nucleic acid sequence that includes one or more nucleotides to be edited, and at least 10, preferably 16 to 40 nucleotides, which is complementary or at least partially complementary to the target sequence in the target RNA; iv) Generate a sequence of a second recruitment portion, which includes a nucleic acid sequence, preferably the sequence defined above, that can bind to a deaminase, preferably adenosine deaminase, without binding to the target RNA; v) Assemble the sequence of the first recruitment portion, the nucleotide spacer, the targeting sequence, and the sequence of the second recruitment portion in the 5' to 3' direction or the 3' to 5' direction, more preferably the 3' to 5' direction; vi) Determine potential intramolecular base pairing events within the assembled artificial nucleic acid and select the artificial nucleic acid that exhibits the smallest potential intramolecular base pairing; vii) Evaluate whether the selected recruitment sequence or cluster of selected recruitment sequences, preferably artificial nucleic acids, can bind to other sites on the target RNA or to other RNA molecules in the organism's transcriptome, resulting in undesirable mismatches and out-of-target events, and eliminate such recruitment sequences or clusters of such recruitment sequences.

[0119] The artificial nucleic acids described herein may be synthesized by methods known in the art based on sequences generated by the methods of the present invention. The artificial nucleic acids may be synthesized chemically from a suitable vector or by in vitro transcription, preferably as described herein. Preferably, the artificial nucleic acids of the present invention are synthesized in vivo from a suitable vector that has been pre-transfected in a cell or organism, as described herein.

[0120] In another aspect, the present invention relates to a data processing device comprising means configured to perform a method for generating a sequence of artificial nucleic acids for site-specific editing of a target RNA, the method being as follows: i) Generate a sequence of a first recruitment region that includes at least one recruitment sequence that binds to a first region in the target RNA; ii) Arbitrarily generate a sequence of nucleotide spacers containing at least one nucleotide; iii) Generate a targeted sequence that includes nucleic acid sequences that are complementary or at least partially complementary to the target sequence in the target RNA containing one or more nucleotides to be edited; iv) generate a sequence of a second recruiting portion capable of recruiting deaminase; and v) Assemble the sequence of the first recruitment portion, optionally a nucleotide spacer, a targeting sequence, and the sequence of the second recruitment portion in the 5' to 3' direction or the 3' to 5' direction.

[0121] Preferably, the data processing device is configured to perform the method of the present invention based on the sequence information of the RNA to be edited and a preset entered by the user with respect to the following points. - Length of the nucleic acid sequence that functions as a binding site to the target sequence on the target RNA, - The distance between the binding site to the target sequence and the binding site to at least one recruitment sequence on the target RNA. - Length of the nucleotide linker, and length of any nucleotide spacer, - The length of a nucleic acid sequence suitable as a binding site to the recruitment sequence on the target RNA, and / or - Distance between binding sites to individual recruitment sequences on target RNA, and candidate artificial nucleic acids for site-specific editing of target RNA according to the present invention.

[0122] In a more preferred embodiment, the data processing device is configured to do the following: vi) Determine the potential intramolecular base pairing events in the assembled artificial nucleic acid and select the artificial nucleic acid that exhibits the least potential intramolecular base pairing; and / or vii) Evaluate whether the selected recruitment sequence or cluster of selected recruitment sequences, preferably artificial nucleic acids, can bind to other sites on the target RNA or to other RNA molecules in the organism's transcriptome, resulting in undesirable mismatches and out-of-target events, and eliminate such recruitment sequences or clusters of such recruitment sequences.

[0123] In a further embodiment, the present invention relates to a computer program that, when the program is executed by a computer, includes instructions causing the computer to perform a method for generating an artificial nucleic acid sequence for site-specific editing of a target RNA, based on the sequence information of the RNA to be edited described above and a preset entered by the user.

[0124] As a basic function, the computer program includes a "Recruitment Cluster Finder" that generates a sequence of recruitment clusters containing several recruitment sequences that make up the recruitment portion of the artificial nucleic acid.

[0125] To achieve this objective, "Recruitment Cluster Finder" can include the following key features: Function 1: This function searches for G, C, T, and GA blocks within the input target sequence.

[0126] [Table 1]

[0127] Function 2: This function recombines recruitment sequences into recruitment clusters. The recruitment clusters are then filtered based on input criteria, recruitment sequence size, and distance. All groups that meet the criteria (hits) are saved by ViennaRNA to a file for later use.

[0128] [Table 2]

[0129] Function 3: This function converts block complements.

[0130] [Table 3]

[0131] Function 4: This function runs ViennaRNA

[0196] to fold the guide RNA. The results are saved in the output list.

[0132] [Table 4]

[0133] Function 5: ViennaRNA results are sorted according to the first selected criterion ("numerical order", "dot / bracket ratio order", or "minimum energy order"). A results table is then generated.

[0134] [Table 5] JPEG0007914954000007.jpg47169

[0135] Furthermore, when the present invention is performed by a computer, the present invention relates to a computer-readable storage medium that includes instructions causing the computer to execute a method for generating a sequence of artificial nucleic acids for site-directed editing of a target RNA. The computer-readable storage medium may, but is not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any suitable combination thereof. More specific examples (non-exclusive list) of computer-readable storage mediums include: electrical connections having one or more wires, portable computer diskettes, hard disks, random access memory (RAM), read-only memory (ROM), erasable and programmable read-only memory (EPROM or flash memory), optical fibers, portable compact disk read-only memory (CD-ROM), optical storage devices, magnetic storage devices, or any suitable combination thereof. A specific example of a computer-readable portable memory device is a USB flash memory device. The computer-readable storage medium may be any tangible medium that can contain or store a program for use by, or in connection with, an instruction execution system, apparatus, or device.

[0136] In one embodiment, the present invention provides a vector encoding an artificial nucleic acid as described herein.

[0137] As used herein, the term “vector” typically refers to a nucleic acid molecule, and preferably to an artificial nucleic acid molecule. In relation to the present invention, a vector is suitable for incorporating or housing a desired nucleic acid sequence (such as a nucleic acid sequence of an artificial nucleic acid or a fragment thereof). Examples of such vectors include storage vectors, expression vectors, cloning vectors, and transfer vectors. Cloning vectors may be, for example, plasmid vectors or bacteriophage vectors. Transfer vectors may be vectors suitable for introducing nucleic acid molecules into cells or organisms, such as viral vectors. Preferably, a vector in the sense of this application includes a cloning site, a selection marker such as an antibiotic resistance factor, and a sequence suitable for vector proliferation, such as an origin of replication.

[0138] The vector may be an RNA vector or a DNA vector. Preferably, the vector is a DNA vector. The vector may be any vector known to those skilled in the art, such as a viral vector or a plasmid vector. Preferably, the vector is a plasmid vector, preferably a DNA plasmid vector. In certain embodiments, the vector is a viral vector, preferably selected from the group consisting of lentiviral vectors, retroviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, and hybrid vectors.

[0139] Preferably, the vector according to the present invention is suitable for the production of artificial nucleic acid molecules, preferably RNA, according to the present invention. Therefore, preferably, the vector contains elements necessary for transcription (such as a promoter (e.g., an RNA polymerase promoter)). Preferably, the vector is suitable for transcription using eukaryotic, prokaryotic, viral, or phage transcription systems, for example, transcription using eukaryotic cells, prokaryotic cells, or eukaryotic, prokaryotic, viral, or phage in vitro transcription systems. Therefore, for example, the vector may contain a promoter sequence that is recognized by a polymerase (such as RNA polymerase), for example, by the RNA polymerase of a eukaryotic, prokaryotic, viral, or phage. In a preferred embodiment, the vector contains a phage RNA polymerase promoter (such as SP6, T3, or T7, preferably a T7 promoter). Preferably, the vector is suitable for in vitro transcription using a phage-based in vitro transcription system, such as an in vitro transcription system based on T7 RNA polymerase.

[0140] In some embodiments, the vector is designed for the transcription of artificial nucleic acids, preferably as described herein, during transfection into eukaryotic cells, preferably into mammalian cells, or during administration to a subject. In preferred embodiments, the vector is designed for the transcription of artificial nucleic acids by eukaryotic RNA polymerase, preferably RNA polymerase II or III, more preferably RNA polymerase III. In certain embodiments, the vector may include a U6 snRNA promoter or an H1 promoter, and optionally, a selection marker, such as a reporter gene (e.g., GFP) or a resistance gene (e.g., a puromycin or hygromycin resistance gene).

[0141] According to one aspect of the present invention, cells comprising the artificial nucleic acid or vector described herein are provided. The cells may be any cells, such as bacterial cells or eukaryotic cells, preferably vertebrate cells such as insect cells, plant cells, or mammalian cells (e.g., human cells or mouse cells). Cells may be used, for example, bacterial cells, for the replication of the vector of the present invention. Furthermore, cells, preferably eukaryotic cells, may be used for the synthesis of the artificial nucleic acid molecule according to the present invention.

[0142] Cells according to the present invention are obtained, for example, by standard nucleic acid introduction methods such as standard transfection, transduction, or transformation methods. As used herein, the term “transfection” generally refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, preferably eukaryotic cells. With respect to the present invention, the term “transfection” encompasses any method known to those skilled in the art for introducing nucleic acid molecules into cells, preferably eukaryotic cells (e.g., mammalian cells). Such methods include, for example, electroporation, lipofection (e.g., lipofection based on cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or cationic polymer-based transfection (e.g., DEAE-dextran or polyethyleneimine). In this regard, the artificial nucleic acids or vectors described herein may be introduced into cells by a transient approach or to maintain the artificial nucleic acids or vectors stably in cells (e.g., in stable cell lines).

[0143] Preferably, the cells are mammalian cells (human target cells, livestock cells, experimental animal cells (mouse cells or rat cells, etc.)). Preferably, the cells are human cells. The cells may be cells from established cell lines such as CHO, BHK, 293T, COS-7, HELA, HEK, or Jarcutt cell lines, or the cells may be primary cells such as human dermal fibroblast (HDF) cells, and preferably cells isolated from living organisms. In a preferred embodiment, the cells are isolated cells of mammalian target, preferably human target.

[0144] In further embodiments, the present invention relates to compositions comprising the artificial nucleic acids, vectors, or cells described herein, and optionally, additional excipients, preferably pharmaceutically acceptable excipients. The compositions described herein are preferably pharmaceutical compositions. The compositions described herein may be used in the treatment or prevention of a target, such as in gene therapy approaches. Alternatively, the compositions may also be used for diagnostic purposes or for experimental use (e.g., in vitro experiments).

[0145] Preferably, the composition further comprises one or more vehicles, diluents, and / or excipients, which are preferably pharmaceutically acceptable. With respect to the present invention, a pharmaceutically acceptable vehicle typically comprises a liquid or non-liquid base for the composition described herein. In one embodiment, the composition is provided in liquid form. In this regard, preferably, the vehicle is water-based, such as water free of pyrogens, isotonic saline, or a buffered (aqueous) solution (e.g., a buffered solution of phosphates, citrates, etc.). The buffer may be hypertonic, isotonic, or hypotonic with respect to a particular reference medium, that is, the buffer may have a higher, the same, or lower salt concentration with respect to a particular reference medium, preferably such a concentration of the salt described above is used that does not cause damage to mammalian cells due to osmotic or other concentration effects. The reference medium is, for example, a liquid produced in vivo (blood, lymph, cytoplasmic fluid, or other body fluids), or, for example, a liquid that can be used as a reference medium in an in vitro method (such as a general buffer or liquid). Such general buffers or liquids are known to those skilled in the art. As the liquid base, Ringer's lactate solution is particularly preferred.

[0146] One or more compatible solid or liquid fillers, diluents, or encapsulating compounds suitable for administration to a target may be used in the pharmaceutical composition of the present invention. As used herein, the term “compatible” preferably means that these components of the (pharmaceutical) composition are miscible with the artificial nucleic acids, vectors, or cells specified herein in such a manner that they do not produce interactions that substantially reduce the pharmaceutically effectiveness of the composition under typical conditions of use.

[0147] The compositions according to the present invention may optionally further contain one or more additional pharmaceutically active ingredients. In this regard, a pharmaceutically active ingredient is a compound that exhibits a therapeutic effect for curing, improving or preventing a particular sign or disease. Such compounds include, but are not limited to, peptides or proteins, nucleic acids, (therapeutably active) low molecular weight organic or inorganic compounds (molecular weight less than 5000, preferably less than 1000), sugars, antigens or antibodies, or other therapeutic agents already known in the prior art.

[0148] Furthermore, the composition may include a carrier for artificial nucleic acid molecules or vectors. Such a carrier may be suitable for solubility in physiologically acceptable liquids, transport and intracellular uptake of pharmaceutically active artificial nucleic acid molecules or vectors. Therefore, such a carrier may be a suitable component for the depot and delivery of the artificial nucleic acid molecules or vectors described herein. Such a component may be, for example, a cationic or polycationic carrier or compound that can function as a transfection agent or complex-forming agent. In this regard, cationic or polycationic compounds are particularly preferred as transfection agents or complex-forming agents.

[0149] The term “cationic compound” typically refers to a positively charged (cationic) molecule with a pH value of 1 to 9, preferably 9 or less (e.g., 5 to 9), or 8 or less (e.g., 5 to 8), or 7 or less (e.g., 5 to 7), most preferably at physiological pH (e.g., 7.3 to 7.4). Therefore, cationic compounds can be selected from any positively charged compound or polymer, preferably a positively charged cationic peptide or protein, or a cationic lipid, under physiological conditions, particularly under physiological conditions in vivo. A “cationic peptide or protein” may contain, for example, at least one positively charged amino acid, or two or more positively charged amino acids, selected from Arg, His, Lys, or Orn. ​​Therefore, “polycationic compounds” also include the range of compounds exhibiting two or more positive charges under given conditions.

[0150] The compositions described herein preferably comprise artificial nucleic acids or vectors in naked or composite form. In preferred embodiments, the compositions comprise artificial nucleic acids or vectors in the form of nanoparticles, preferably lipid nanoparticles or liposomes.

[0151] In a further embodiment, the present invention relates to a kit or kit of parts comprising the artificial nucleic acid molecule, vector, cell and / or (pharmaceutical) composition of the present invention.

[0152] Preferably, the kit further comprises instructions for use, cells for transfection, means for administering the composition, a (pharmaceutically acceptable) carrier or vehicle, and / or a (pharmaceutically acceptable) solution for dissolving or diluting the artificial nucleic acid molecule, vector, cells, or composition. In a preferred embodiment, the kit comprises the artificial nucleic acid or vector described herein in either liquid or solid form (e.g., lyophilized), and a (pharmaceutically acceptable) vehicle for administration. For example, the kit may comprise the artificial nucleic acid or vector and the vehicle (e.g., water, PBS, Ringer's lactate, or other suitable buffer), which are mixed before administration to the subject.

[0153] In a further embodiment, the present invention relates to the use of the artificial nucleic acids, vectors, cells, compositions, or kits described herein.

[0154] In particular, the present invention includes the use of artificial nucleic acids, vectors, cells, compositions, or kits for site-directed editing of target RNA. Therefore, the artificial nucleic acids, vectors, cells, compositions, or kits described herein are preferably used to facilitate site-directed editing of target RNA by specifically binding to the target RNA via a targeting sequence and by recruiting at least one recruitment sequence, thereby a deaminase described herein, to the target site. This reaction may occur in vitro or in vivo.

[0155] In a preferred embodiment, the artificial nucleic acid, vector, or composition is administered to or introduced into cells containing the target RNA to be edited. The cells containing the target RNA preferably further contain a deaminase as described herein. The deaminase is preferably an endogenous deaminase, more preferably an adenosine deaminase or cytidine deaminase, or a recombinant deaminase (preferably such as a tagged deaminase or mutant deaminase as described herein), which is preferably stably expressed in or introduced into the cells prior to or simultaneously with the artificial nucleic acid, vector, or composition. Alternatively, the cells containing the artificial nucleic acid or vector as described herein are preferably used for site-directed editing of the target RNA by, for example, transfection, bringing the cells into contact with the target RNA, or by introducing the target RNA into the cells, as described herein.

[0156] In a more preferred embodiment, the present invention provides a method for site-directly editing a target RNA, comprising contacting the target RNA with an artificial nucleic acid, and essentially comprising the steps described herein relating to the use of artificial nucleic acids, vectors, compositions, or cells for site-directly editing RNA.

[0157] The editing reaction is preferably monitored or controlled by sequence analysis of the target RNA.

[0158] The uses and methods described herein may also be employed in the in vitro diagnosis of a disease or disorder. Here, the disease or disorder is preferably selected from the group consisting of infectious diseases, neoplastic diseases, cardiovascular diseases, autoimmune diseases, allergies, and neurological diseases or disorders, and more preferably selected from hereditary diseases or disorders, which are preferably selected from the group consisting of metabolic diseases, neoplastic diseases, autoimmune diseases, cardiovascular diseases, and neurological diseases.

[0159] In further embodiments, the artificial nucleic acids, vectors, cells, compositions, or kits described herein are provided for use as pharmaceuticals in gene therapy, for example. Preferably, the artificial nucleic acids, vectors, compositions, cells, or kits described herein are provided for use in the treatment or prevention of a disease or disorder, wherein the disease or disorder is selected from the group consisting of infectious diseases, neoplastic diseases, cardiovascular diseases, autoimmune diseases, allergies, and neurological diseases or disorders. According to preferred embodiments, the artificial nucleic acids, vectors, cells, compositions, or kits described herein are provided for use as pharmaceuticals or for use in the treatment or prevention of a disease or disorder (preferably as defined herein), wherein the use as a pharmaceutical or treatment or prevention includes a site-directed editing step of a target RNA.

[0160] In one embodiment, the present invention provides a method for treating a subject suffering from a disease or disorder, the method comprising the step of administering an effective amount of an artificial nucleic acid, vector, cell or composition described herein to the subject. The effective amount as relating to this disclosure is typically understood to be an amount sufficient to induce a desired therapeutic effect, i.e., an amount sufficient to achieve editing of a target RNA.

[0161] The disease or disorder may be selected from the group consisting of infectious diseases, neoplastic diseases, cardiovascular diseases, autoimmune diseases, allergies, and neurological diseases or disorders, wherein the disease or disorder is preferably selected from hereditary diseases or hereditary disorders, and preferably selected from the group consisting of metabolic diseases, neoplastic diseases, autoimmune diseases, cardiovascular diseases, and neurological diseases.

[0162] The artificial nucleic acids, vectors, cells, or (pharmaceutical) compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, transvaginally, via an implanted reservoir, or by jet injection. As used herein, "parenteral" includes techniques such as intravitreous, subretinal, subcutaneous, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, and sublingual injection or infusion. In preferred embodiments, the artificial nucleic acid molecules, vectors, cells, or (pharmaceutical) compositions described herein are administered by needle-free injection (e.g., jet injection).

[0163] Preferably, the artificial nucleic acids, vectors, cells, or (pharmaceutical) compositions described herein are administered parenterally, for example by injection, and more preferably by techniques of intravitreous, subretinal, subcutaneous, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intra-arterial, sublingual injection, or infusion. Intradermal and intramuscular injections are particularly preferred. The injectable sterile forms of the pharmaceutical compositions of the present invention may be aqueous suspensions or oily suspensions. These suspensions may be prepared according to techniques known in the art using suitable dispersants or wetting agents and suspending agents.

[0164] The artificial nucleic acids, vectors, cells, or (pharmaceutical) compositions described herein may also be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, or solutions.

[0165] The artificial nucleic acids, vectors, cells, or (pharmaceutical) compositions described herein may also be administered topically, particularly when the therapeutic target includes areas or organs that are easily accessible by topical administration, such as diseases of the skin or any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical administration, the artificial nucleic acids, vectors, cells, or (pharmaceutical) compositions described herein may be formulated in suitable ointments suspended or dissolved on one or more carriers.

[0166] In one embodiment, use as a pharmaceutical includes a process of transfection of mammalian cells, preferably an in vitro or ex vivo transfection of mammalian cells, more preferably an in vitro transfection of isolated cells of the subject to be treated by the pharmaceutical. If the use includes in vitro transfection of isolated cells, use as a pharmaceutical may further include readmission of the transfected cells to the patient. Use as a pharmaceutical of artificial nucleic acids or vectors may further include a selection process of successfully transfected isolated cells. Thus, vectors may be beneficial if they further include selection markers.

[0167] According to another aspect of the present invention, the artificial nucleic acids, vectors, cells, or (pharmaceutical) compositions described herein are provided for use in the diagnosis of a disease or disorder, where the disease or disorder is preferably selected from the group consisting of infectious diseases, neoplastic diseases, cardiovascular diseases, autoimmune diseases, allergies, and neurological diseases or disorders, and in particular selected from hereditary diseases or disorders, preferably selected from the group consisting of metabolic diseases, neoplastic diseases, autoimmune diseases, cardiovascular diseases, and neurological diseases.

[0168] [Brief explanation of the drawing] The following drawings are for illustrative purposes only and further illustrate the present invention. These drawings should not be construed as limiting the present invention.

[0169] Figure 1: Artificial nucleic acids used in Example 1: A: An artificial nucleic acid molecule comprising a 20-nucleotide antisense moiety with a C / A mismatch at position 8, and a schematic R / G motif containing a stem-loop structure.

[0170] B: An artificial nucleic acid molecule according to the present invention, comprising a 20-nucleotide targeting sequence (TS) having a C / A mismatch at position 8, an R / G motif having a stem-loop structure, and a cluster of three recruitment sequences (RS #1, RS #2, RS #3) having 11 to 16 nucleotides linked via an adenosine linker (AAA).

[0171] B: Editing of a dual luciferase W417X amber reporter using endogenous ADAR1 in HeLa cells. 120,000 cells were seeded in 24-well scale. 24 hours after seeding, cells were transfected per well with 800 ng of plasmids encoding artificial nucleic acids A and B, respectively, and 200 ng of a dual luciferase reporter, using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. 72 hours after transfection, cells were harvested. RNA isolation, DNase-I digestion, and RT-PCR were followed by Sanger sequencing.

[0172] Figure 2: A: Artificial nucleic acids and R / G motif versions used in Example 2: (A): A prior art design that lacks a recruitment array (RS) and has a 16nt antisense portion and an R / G motif version 20 (RG-V20).

[0173] (B): A novel design with a recruitment cluster including three recruitment sequences (3×RS) and R / G motif version 21 (RG-V21).

[0174] (C): RG motifs version 20 and version 21 array.

[0175] B: Editing in HeLa cells using endogenous human ADAR1 and adenovirus (AdV) encoded R / G gRNA.

[0176] 200k HeLa cells were seeded in a 96-well scale. Reverse transduction was performed at seeding using 175 MOI gRNA AdV. Forward transduction was achieved 24 hours after seeding using 175 MOI dual luciferase wt / amb AdV or 5 MOI wt / wt. Note: Firefly is always normalized on Renilla, so the amount of wt / wt AdV does not affect the final percentage. Luciferase assays were performed 96 hours after reverse infection.

[0177] Figure 3: Editing of dual luciferase reporters via recruitment of endogenous human ADAR1 using AdV-encoding 3× RS guide RNA in several cell lines. Analysis via dual luciferase assay (N=3, each with 2 technical replicates).

[0178] 25,000 cells of each cell type were seeded in a 96-well scale. In HeLa cells, forward transduction was achieved 24 hours after seeding. In all other cell types, reverse transduction was achieved at seeding. Collection and luciferase assays were performed 96 hours after infection.

[0179] Luciferase assay: 420 datapoints (35 settings, 6 Renira and 6 Fireflies per setting) Settings: gRNA MOI: HeLa(100 MOI), SK-N-BE(100 MOI), Huh7(75 MOI), A549(75 MOI), HepG2(75 MOI), SY5Y(100 MOI), U87MG(100 MOI), U2OS(75 MOI);DL wt / amb 50 MOI;DL wt / wt 5 M.O.I.

[0180] Figure 4: Editing of dual luciferase reporters via recruitment of endogenous human ADAR1 using AdV-encoding 3× RS guide RNA in several cell lines. Analysis via Sanger sequencing.

[0181] For Huh7 cells, SK-N-BE cells, A549 cells, HepG2 cells, and SY5Y cells: 25,000 cells per well (96-well scale) were reverse-infected at the MOI indicated on day 1 seeding. The culture medium was changed on days 2-4. On day 5, 6 wells were collected and pooled per RT-PCR cycle. For HeLa cells: 25,000 cells per well (96-well scale) were seeded on day 1. On day 2, forward infection was performed using the following MOIs: HeLa: n=2 [75 MOI], n=2 [100 MOI], n=2 [125 MOI]; Huh7: n=3 [75 MOI]; A549: n=3 [75 MOI]; HepG2: n=3 [75 MOI]; SK-N-BE(2): n=3 [100 MOI]; SH-SY5Y: n=3 [100 MOI]. The culture medium was changed on days 3-5. On day 6, 6 wells were collected and pooled per RT-PCR cycle.

[0182] Figure 5: A: Editing of several disease-associated target mRNAs in HeLa cells using 6-9× RS guide RNA and endogenous ADAR1. 120,000 cells were seeded in 24-well scale. 24 hours after seeding, cells were transfected with 800 ng of guide RNA plasmid and 200 ng of target coding plasmid (cDNA) per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. 72 hours after transfection, cells were harvested. RNA isolation, DNase-I digestion, and RT-PCR were followed by Sanger sequencing.

[0183] B: An exemplary Sanger sequence trace of the data shown in Figure 5A, demonstrating the complete absence of off-target bystander editing around the target site.

[0184] Figure 6: Plasmid-encoding RG-V21_20p8_3×RS and RG-V21_20p8_8×RS guide RNAs primarily recruit ADAR1 isoform p110 in HeLa cells. 1.2×10 5HeLa cells were seeded in a 12-well scale, and reverse transfection was achieved with scrambled siRNA, ADAR1 siRNA, or ADAR1p150 siRNA using 2.5 pmol siRNA per well (3 μl HighPerfect + 2.5 μl 1 μM siRNA, and 200 μl OptiMem). Each siRNA was seeded in 6 wells. Each well used in the 12-well plate contained the indicated 200 μl transfection mixture for the corresponding siRNA. 24 hours after siRNA transfection, similarly treated wells were harvested and the cells were pooled. 25,000 differently treated HeLa cells were then seeded in a 96-well scale. 24 hours after seeding, the cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0185] Figure 7: Effect of recruitment sequence number on editing yield. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. The location of target sequences on the luciferase transcript is shown in dark gray, and the location of each RS binding region is shown in light gray. Editing target

[0186] [ka]

[0187] The positions are also shown. Each RS is 9-16 nt long and is separated by an AAA linker.

[0188] Figure 8: A: Effect of recruitment sequence length (7-15 nt / RS) on editing yield. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0189] B: Schematic diagram of the minimum free energy structure of guide RNA at 37°C.

[0190] Figure 9: A: Effect of adenosine linker length on editing yield. Luciferase activity (multiplier change) is shown next to a schematic diagram indicating the corresponding gRNA-binding region (BR) on mRNA. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0191] B: The dataset in Figure 9A is shown next to a schematic diagram illustrating the corresponding gRNA composition with respect to the number of adenosine nucleotides used as linkers between recruitment sequences (RSs) and the number of adenosine nucleotides used as spacers between recruitment sequence #1 and the target sequence.

[0192] Figure 10: Optimization of the arrangement of three recruitment sequences on target mRNA. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. (S = short distance between two RSs (15-62 nt); L = long distance between two RSs (375-460 nt)) Figure 11: Optimization of the placement of an extended recruitment sequence (20 nt) in the context of two short recruitment sequences (15 nt) on target mRNA. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. (S = short distance between two RSs (15-62 nt); L = long distance between two RSs (375-460 nt); RSs extended from 15 nt to 20 nt are highlighted) Figure 12:A: Effect of recruitment sequence masking (simulation of guide RNA with strong secondary structure in the antisense region). The upper left portion of Figure 12A depicts the representation used to show gRNA-mRNA interaction. The binding schematic portion shows the binding region (BR) of the gRNA recruitment sequence on the mRNA. RS#1 binds to BR#1. The gRNA schematic shows the gRNA composition consisting of the R / G motif V21, targeting sequence, adenosine nucleotide linker / spacer, recruitment sequences (RS)#1-#3, and in some cases, masking recruitment sequences (mRS)#4-#6. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. Twenty-four hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0193] B: Schematic diagram of the minimum free energy structure of guide RNA at 37°C.

[0194] Figure 13: Editing of the dual luciferase W417X amber reporter in C57BL / 6 mice after hydrodynamic tail vein (HDTV) injection of gRNA and reporter plasmid. Negative control mice were treated with 10 μg of dual luciferase wt / amb reporter plasmid, positive control mice were treated with 10 μg of dual luciferase wt / wt reporter plasmid, and edited mice were treated with 5 μg of dual luciferase wt / amb reporter plasmid and 25 μg of guide RNA plasmid. The guide RNA was a 20-15-15-20p8 guide RNA, and therefore contained three RSs (having lengths of 20 nt, 15 nt, and 15 nt) and a typical 20 nt targeting sequence with adenosine at position 8 and cytosine mismatched. The guide RNA further contained a V21R / G motif at its 5' end (not shown). The edits were analyzed by dual luciferase assay (protein level) and by Sanger sequencing after reverse transcription (RNA level). Exemplary Sanger sequence traces are shown.

[0195] Figure 14: Editing of endogenous UTR target using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene 6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added and PCR was performed. This was followed by 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG). Exemplary Sanger sequencing traces are shown, indicating the editing sites and yields. "A" and "G" represent the adenosine peak and the guanosine peak, respectively.

[0196] Figure 15: Editing of endogenous ORF targets using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene 6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added and PCR was performed. This was followed by 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG). Exemplary Sanger sequencing traces are shown, indicating the editing sites and editing yields. "A" and "G" represent the adenosine peak and the guanosine peak, respectively.

[0197] Figure 16: A: A benchmark comparing the guide RNA of the present invention with a conventional LEAPER gRNA targeting NUP43 V233V. Editing of endogenous targets using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene 6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added and PCR was performed. Following this, 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG) were performed. N=3 biological replicates.

[0198] B: A benchmark comparing the guide RNA of the present invention with a conventional LEAPER gRNA targeting RAB7A 3'UTR TAG#1. Editing of endogenous targets using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added and PCR was performed. Following this, 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG) were performed. N=3 biological replicates.

[0199] Figure 17: Recruitment cluster in silico optimization using the Recruitment Cluster Finder tool.

[0200] Figure 18:A: Legend for Figures 18B and 18C. RM = Recruitment portion.

[0201] B: The recruitment sequence (RS) and double-stranded ADAR recruitment domain (e.g., R / G motif) can be positioned around the targeting sequence (TS) with certain flexibility. The components of the antisense portion of the CLUSTER guide RNA targeting a dual luciferase reporter in HeLa cells were newly positioned, starting from the conventional #6-#5-#4-TS design.

[0202] C: Results of the dual luciferase assay. Data are shown as mean ± sd of N=5 biological replicates. In particular, several designs (e.g., #4-TS-#3-#2) gave better editing yields compared to the conventional #6-#5-#4-TS design. Importantly, the former design allows for the inclusion of the sequence space 3' of targeted adenosine for binding to the CLUSTER guide RNA (e.g., binding sites #3, #2, #1). Luciferase assay setup for this experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid vs. Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0203] Figure 19: A: Correction of disease-related hIDUA W402X Amber mutation in primary fibroblasts (Hurler FB, GM06214) derived from patients with Hurler syndrome by transfection with chemically synthesized CLUSTER antisense oligonucleotides (ASOs). Edit yields were determined by Sanger sequencing. Fibroblasts derived from patients with Hurler syndrome (GM06214) were purchased from the Coriel Institute for Medical Research (USA). 2.5 × 10⁶ cells in 2.5 ml of DMEM + 15% FBS 5Cells / well were seeded in a 6-well plate. For each test condition, RNA editing rate was determined by Sanger sequencing using one 6-well. CLUSTER ASO is a PAGE-purified, terminally blocked (2'-OMe,PS)RNA oligonucleotide with a 3×RS(20-20p8-25-20)CLUSTER design, which was ligated in-house (T4 RNA ligase) according to our recently published protocol from two commercially purchased (Biospring GmbH, Germany), 69nt (5' portion) and 80nt (3' portion) HPLC-purified oligonucleotides. The complete sequences and modification patterns are shown in the following list of applied gRNAs. Transfection was performed 24 hours after seeding with 125 pmol of ASO and 7.5 μl of RNAiMAX, respectively, diluted in 250 μl of Opti-MEM. Both solutions were incubated for 5 minutes, then combined and incubated for another 20 minutes before the transfection mixture was evenly distributed into one well. The culture medium was changed 24 hours after transfection. 48 hours after transfection, fibroblasts were harvested in RLT buffer (QIAGEN, #79216) followed by RNA isolation using the Monarch RNA Cleanup Kit (NEB, #T2030L). Turbo-DNase digestion was performed using the TURBO DNA-free Kit (Thermofisher, #AM1907). Reverse transcription was performed using SuperScript IV RT (Thermofisher, #18090050) with random primers at 52°C for 1 hour. Standard PCR followed by nested PCR was performed using Taq polymerase (NEB, #M267S) in each reaction containing 10% DMSO. After the second PCR, the products were separated by SB-agarose gel electrophoresis. After PCR cleanup (NucleoSpin Gel and PCR Clean-up Kit, Macherey Nagel, #740609), Sanger sequencing (Microsynth AG) was performed.

[0204] B: Furthermore, the restoration of protein function was determined using an α-L-iduronidase enzyme activity assay. The hIDUA enzyme activity measured in Hurler fibroblasts was normalized to the IDUA enzyme activity measured in Scheie patient fibroblasts (Scheie FB, GM01323). Data are shown as the mean ± sd of N=4-5 biological replicates, as indicated by individual data points. Patient-derived fibroblasts from Scheie syndrome (GM01323) and Hurler syndrome (GM06214) were purchased from the Coriel Institute for Medical Research (USA). 2.5 × 10⁶ cells in 2.5 ml of DMEM + 15% FBS 5Cells / well were seeded in 6-well plates. For each test condition, two 6-well plates were used for the IDUA assay. CLUSTER ASO was a PAGE-purified, terminally blocked (2'-OMe,PS)RNA oligonucleotide with a 3×RS(20-20p8-25-20)CLUSTER design, which was ligated in-house (T4 RNA ligase) according to our recently published protocol from two commercially available products (Biospring GmbH, Germany) as well as HPLC-purified oligonucleotides of 69nt (5' portion) and 80nt (3' portion) lengths. The complete sequence and modification patterns are shown in the following list of applied gRNAs. Transfection was performed 24 hours after seeding with 125 pmol of ASO and 7.5 μl of RNAiMAX, each diluted in 250 μl of Opti-MEM. After incubating both solutions for 5 minutes, they were combined and incubated for a further 20 minutes before the transfection mixture was evenly distributed into one well. The culture medium was changed 24 hours after transfection. 48 hours after transfection, fibroblasts were isolated and washed once with PBS. 40 μl of 0.5% Triton X-100 in PBS was added to the cell pellet, incubated on ice for 30 minutes, and performed an α-L-iduronidase enzyme assay. Standard dilutions of 4-methylumbelliferone (Sigma Aldrich, M1381) were prepared in 1×PBS for edit readout by the α-L-iduronidase enzyme activity assay. For each concentration, 25 μl of the standard solution was added to 25 μl of 0.4 M sodium formate buffer (pH 3.5), and applied in 3 replicates to a 96-well LumiNunc plate (VWR, 732-2696). The substrate (4-methylumbelliferyl α-L-idulonide, glycosine, #44076) was dissolved in 0.4 M sodium formate buffer to a final concentration of 180 μM. For the mouse IDUA assay using HeLa cells, 25 μl of 1:3 diluted cell lysate (0.5% Tween-20 / PBS) was added to 25 μl of substrate in a plate, and incubated in the dark at 37°C for 45 minutes.25 μl of undiluted cell lysate (0.5% Triton X-100 / PBS) was added to 25 μl of substrate in a plate and incubated in the dark at 37°C for 90 minutes. The reaction was quenched in both cases by adding 200 μl of glycine carbonate buffer (0.17 M glycine / NaOH, pH 10.4). The fluorescence of 4-methylumbelliferone was measured using a Tecan Spark 10M plate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The calculated enzyme activity was based on the protein amount determined by a BCA assay (Pierce BCA Protein Assay Kit, Thermofisher, 23227). Enzyme activity was normalized to Scheie fibroblast lysate.

[0205] Figure 20: A: 19-11-13-20p8 Analysis of off-target editing in the poly(A)+ transcriptome to recruit endogenous ADARs from 293 FT cells to the 5'UAG site in the 3'-UTR of endogenous RAB7A using CLUSTER guide RNA. The scatter plot shows differential editing at approximately 30,000 sites, comparing editing levels in cells transfected with plasmids containing CLUSTER or non-targeting guide RNA. Experiments were performed using two independent replicates. Edits on targets are indicated by arrows. Significantly different editing sites (adjusted P<0.01, Fisher's exact test, two-tailed, N≧50) are highlighted in black. Using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format, 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) was added 24 hours after seeding to 6 × 10⁶ cells. 4RNA editing was performed by transfection of individual HEK293FT cells. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included untargeted guide RNA (NT-RNA) and RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA. RNA was isolated using the RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and re-purified using the RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. The library was prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq6000 (50M reads, 2×100bp paired-end, Illumina, USA).

[0206] B: Analysis of off-target editing in the poly(A)+ transcriptome to recruit endogenous ADAR to the 5'UAG site in the 3'-UTR of endogenous RAB7A with 111nt LEAPER guide RNA from 293 FT cells. The scatter plot shows differential editing at approximately 30,000 sites comparing editing levels in cells transfected with plasmids containing LEAPER or non-targeted guide RNA. Experiments were performed using two independent replicates. Edits on targets are indicated by arrows. Significantly different editing sites (adjusted P<0.01, exact test, two-sided, N≧50) are highlighted in black. 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) was used with FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format, 24 hours after seeding. 4RNA editing was performed by transfection of individual HEK293FT cells. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included untargeted guide RNA (NT-RNA) and RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated with RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again with RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. The library was prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq6000 (50M reads, 2×100bp paired-end, Illumina, USA).

[0207] C: Editing accuracy was evaluated by analyzing all NGS reads with targeted editing for bystander editing. CLUSTER guide RNA gave mainly clean sequencing reads and reads with a small percentage of single bystander edits, while reads from LEAPER samples very frequently contained several bystander edits. FuGene6 (Promega, #E2691) was used in a 1:3 ratio in a 24-cell format, and 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) was seeded 24 hours later in 6 × 10⁶ cells. 4RNA editing was performed by transfection of individual HEK293FT cells. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included (1) untargeted guide RNA (NT-RNA), (2) RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA, and (3) RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated with RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again with RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. The library was prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA).

[0208] D: Estimation of yield of clean edits (no bystander) and all edits on the target. Using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format, 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) was seeded 24 hours later in 6 × 10⁶ cells. 4RNA editing was performed by transfection of individual HEK293FT cells. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included (1) untargeted guide RNA (NT-RNA), (2) RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA, and (3) RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated with RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again with RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. The library was prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA).

[0209] Figure 21: List of off-target events induced by RNA editing using CLUSTER guide RNA and LEAPER guide RNA, as determined by next-generation sequencing. This list shows a subset of off-target editing events at “unknown” sites detected by a pipeline that searches for sites edited to be significantly distinguishable in at least one sample when cells are treated with either CLUSTER guide RNA or LEAPER guide RNA and compared to a non-targeting control guide RNA (NT gRNA). Sites were assigned “unknown” if they were not enumerated in the RADAR database. Non-synonymous editing was detected only for a single site, HTATSF1(S742G). Mapping analysis detected sites for potential off-target binding of RAB7A guide RNA to CTNNAL1, HTATSF1, and ZNF740. Detected RAB7A sites represent the proportion of bystander editing sites close to the target site that meet the pipeline's significance and cutoff criteria. Data represent the mean ± sd of N=2 NGS repeats. Using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format, 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) was added 24 hours after seeding to 6 × 10⁶ cells. 4RNA editing was performed by transfection of individual HEK293FT cells. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included (1) untargeted guide RNA (NT-RNA), (2) RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA, and (3) RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated with RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again with RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. The library was prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA).

[0210] Examples The following embodiments are merely illustrative and further illustrate the present invention. These embodiments should not be construed as limiting the present invention.

[0211] Unless otherwise stated, nucleic acid sequences provided herein are printed from 5' to 3'. In other words, unless otherwise stated, the first nucleotide residue in a nucleic acid sequence printed herein is the 5' end of the nucleic acid sequence. Unless otherwise stated, amino acid sequences are printed from the N-terminus to the C-terminus.

[0212] Example 1: Editing of a dual luciferase W417X amber reporter using endogenous ADAR1 in HeLa cells The general structure of the artificial nucleic acid used in Example 1 is shown in Figure 1A: A: An artificial nucleic acid molecule from prior art, comprising an antisense moiety of 20 nucleotides having a C / A mismatch at position 8, and a schematic R / G motif having a stem-loop structure.

[0213] B: An artificial nucleic acid molecule according to the present invention, comprising an antisense moiety of 20 nucleotides having a C / A mismatch at position 8, an R / G motif having a stem-loop structure, and a cluster of three recruitment sequences (RS #1, RS #2, RS #3) having 11 to 16 nucleotides linked via an adenosine linker (AAA).

[0214] 120,000 HeLa cells were seeded in 24-well scales. 24 hours after seeding, cells were transfected per well with 800 ng of RNA plasmids encoding artificial nucleic acids A and B, respectively, as well as artificial nucleic acids containing a 40 nt antisense moiety, and artificial nucleic acids containing a cluster of 8 recruitment sequences, and 200 ng of a dual luciferase reporter, using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. 72 hours after transfection, cells were harvested. RNA isolation, DNase-I digestion, and RT-PCR were followed by Sanger sequencing.

[0215] As shown in Figure 1B, the prior art 20nt short R / G guide RNA without a recruitment sequence (RS) cannot efficiently recruit endogenous ADAR1 in HeLa cells. Adding three recruitment sequences to a guide RNA with a 20nt targeting sequence dramatically increases editing from 0% to 20%. Adding eight recruitment sequences to a guide RNA with a 20nt targeting sequence dramatically increases editing from 0% to 24%. Treatment of HeLa cells with IFNa slightly increases editing.

[0216] Example 2: Editing in HeLa cells using endogenous human ADAR1 and AdV-coding R / G gRNA In further experiments, RNA editing in HeLa cells was studied using a prior art guide RNA design comprising an R / G-V20 motif and a 16-nucleotide targeting sequence, and an artificial nucleic acid according to the present invention comprising an R / G-V21 motif, a 20-nucleotide targeting sequence, and a recruitment cluster containing three recruitment sequences having 11-16 nucleotides linked by a triple adenosine linker (AAA). Note that the regions on the target RNA bound by the recruitment sequences (RS #1, RS #2, RS #3) of the artificial nucleic acid are separated by 10-100 nucleotides on the target RNA (see Figure 2A).

[0217] 200k HeLa cells were seeded in a 96-well scale. Reverse transduction was performed at seeding using 175 MOI (multiple degree of infection) gRNA AdV. Forward transduction was achieved 24 hours after seeding using 175 MOI dual luciferase wt / amb AdV or 5 MOI wt / wt. Luciferase assays were performed 96 hours after reverse infection.

[0218] As shown in Figure 2B, the artificial nucleic acid according to the present invention, which includes a recruitment moiety containing three recruitment sequences (RS #1, RS #2, RS #3) that bind to a region of 11-16 nucleotides on the target RNA, restores luciferase activity to 100%, whereas the prior art design lacking the recruitment sequences can only restore (normalized) luciferase activity to 1.5%.

[0219] Example 3: Editing of a dual luciferase reporter via recruitment of endogenous human ADAR1 using adenovirus-encoded 3× RS guide RNA in several cell lines. To evaluate the editing of a dual luciferase reporter via recruitment of endogenous human ADAR1 using adenovirus (AdV)-encoding 3× RS guide RNA in several cell lines, 25k cells of each cell type were seeded in 96-well scales. In HeLa cells, forward transduction was achieved 24 hours after seeding. In all other cell types, reverse transduction was achieved at seeding. Collection and luciferase assays were performed 96 hours after infection.

[0220] As shown in Figure 3, restoration of firefly luciferase activity by RNA editing was achieved in several cell lines derived from different tissues (brain, liver, lung, and cervix) by utilizing only endogenous ADAR1. In HeLa cells, normalized firefly luciferase activity could be restored to 90% of wild-type levels. Readout was performed using a luciferase assay.

[0221] Example 4: Editing of dual luciferase reporters in several cell lines using AdV-encoding 3× RS guide RNA and endogenous ADAR1 For Huh7, SK-N-BE, A549, HepG2, and SY5Y cells: 25,000 cells per well (96-well scale) were reverse-infected at the indicated MOI at the time of seeding on day 1. The culture medium was changed on days 2-4. Six wells were collected and pooled per RT-PCR cycle on day 5. For HeLa cells: 25,000 cells per well (96-well scale) were seeded on day 1. Forward infection at the indicated MOI was performed on day 2. The culture medium was changed on days 3-5. Six wells were collected and pooled per RT-PCR cycle on day 6.

[0222] As shown in Figure 4, editing could be achieved in several cell lines derived from different tissues (brain, liver, lung, and cervix) by using only endogenous ADAR1. In this case, readout was performed by Sanger sequencing (RNA level). However, the results were consistent with the recovery of luciferase activity (protein level) shown in Example 3, Figure 3.

[0223] Example 5: Editing of several disease-related target mRNAs in HeLa cells using 6-9×RS guide RNA and endogenous ADAR1 To evaluate the therapeutic potential of the artificial nucleic acid (guide RNA) of the present invention, editing of the following disease-related target mRNAs in HeLa cells was tested using 6-9×RS guide RNA and endogenous ADAR1. The target mRNAs were encoded as cDNAs on a pcDNA3 expression plasmid.

[0224] Mutations in BMPR2, the bone morphogenetic protein receptor type II (BMPR2), are the most common genetic cause of pulmonary hypertension.

[0225] Mutations in COL3A1 have been identified as underlying Ehlers-Danloe syndrome type IV, an autosomal dominant connective tissue disorder.

[0226] Diseases associated with mutations in FANCC (Fanconi anemia complementation group C) include Fanconi anemia, complementation group C, and Fanconi anemia, complementation group A.

[0227] AHI: AHI1 specifically encodes the Jouberin protein, and mutations in the expression of this gene are known to cause specific forms of Joubert syndrome.

[0228] Mutations in MYBPC (cardiac myosin-binding protein C, MyBP-C, encoded by MYBPC3) are the most common cause of hypertrophic cardiomyopathy.

[0229] IL2RG: Severe combined immunodeficiency (SCID) is a syndrome of severe cellular and humoral immune deficiencies. In humans, SCID is most commonly caused by mutations in the X-linked gene IL2RG, which encodes γc, the common gamma chain of leukocyte receptors for interleukin-2 and several other cytokines.

[0230] PINK1:PTEN-induced kinase 1 (PINK1) is a mitochondrial serine / threonine-protein kinase encoded by the PINK1 gene. It is thought to protect cells from stress-induced mitochondrial dysfunction. Mutations in this gene cause a form of autosomal recessive early-onset Parkinson's disease.

[0231] 120,000 cells were seeded in 24-well scales. 24 hours after seeding, cells were transfected with 800 ng of guide RNA plasmid and 200 ng of target coding plasmid (cDNA) per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. 72 hours after transfection, cells were harvested. After RNA isolation, DNase-I digestion and RT-PCR were performed, followed by Sanger sequencing.

[0232] As shown in Figure 5A, editing at the target site was detected in all disease-associated target mRNAs. The level of editing at the target varied from 5–15% for PINK1_W437X, IL2RG_W237X, and MYBPC3_W1098X mRNA, up to 30% for AHI_W725X and FANCC_W506X mRNA, and up to 55–60% for COL3A1_W1278X and BMPR2_W298X mRNA. In particular, off-target bystander editing was not detected by Sanger sequencing, even at editable adenosine nucleotides located very close to the target adenosine. Four exemplary Sanger sequence traces illustrating this are shown in Figure 5B.

[0233] Example 6: RG-V21_20p8_3×RS guide RNA-encoding plasmid and RG-V21_20p8_8×RS guide RNA-encoding plasmid mainly recruit ADAR1p110 in HeLa cells To investigate which ADAR isoform is mainly recruited by the artificial nucleic acid (guide RNA) of the present invention, 1.2×10 5 HeLa cells were seeded in 12-well scale, and reverse transfection was performed using scrambled siRNA, ADAR1 siRNA, or ADAR1p150 siRNA with 2.5 pmol of siRNA per well (3 μl of HighPerfect + 2.5 μl of 1 μM siRNA, 200 μl using OptiMem). Seeding was performed in 6 wells per siRNA. Each used well of the 12-well plate contained 200 μl of the indicated transfection mixture for the corresponding siRNA. 24 hours after siRNA transfection, similarly treated wells were harvested and cells were pooled. Then, 25,000 separately treated HeLa cells were seeded in 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual-luciferase reporter per well using a plasmid to Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual-luciferase reporter assay system.

[0234] As shown in Figure 6, siRNA knockdown of ADAR1 (isoforms p110 and p150) reduced editing by nearly 10-fold in the luciferase assay, while specific ADAR1p150 knockdown had only a minimal effect on editing yield. Interferon-α induction of cells did not significantly increase editing yield, supporting the idea that editing is carried out by constitutively expressed ADAR1p110 rather than by the IFN-α-inducible ADAR1p150 isoform. As seen in the corresponding Western blots, the knockdown was highly efficient, resulting in almost complete removal of the target protein.

[0235] Example 7: Effect of the number of recruitment sequences on editing yield To investigate the effect of the number of recruitment sequences in the recruitment region on the editing yield, several artificial nucleic acids (guide RNAs) were constructed, each containing 0, 1, 2, 3, 6, 8, or 20 recruitment sequences, a 20nt targeting sequence, and an RG-V21 motif. The RS sequences ranged in length from 9 to 16nt and were linked together by triple adenosine linkers.

[0236] 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0237] As shown in Figure 7, the number of RS elements in this invention is important for achieving efficient editing. At least two RS elements provide significant editing. Three to eight RS elements appear to be optimal. However, simply increasing the number of RS elements, e.g., 20 × RS, did not result in an improved editing yield.

[0238] Example 8: Effect of recruitment sequence length on editing yield To investigate the effect of recruitment sequence length on editing yield, several artificial nucleic acids (guide RNAs) according to the present invention were constructed, each having a recruitment region containing three recruitment sequences of different lengths. The lengths of the RS sequences and the locations of their binding regions on the target mRNA are shown in Figure 8 for different constructs. Target sequence, RS binding region, and target codon

[0239] [ka]

[0240] This indicates that the guide RNA also contained a 5' terminal R / G motif (V21, not shown).

[0241] 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0242] As shown in Figure 8, the length of the recruitment sequence (RS) is an important optimization parameter in this invention for achieving efficient editing. Typically, a significant editing yield is obtained using a recruitment sequence (RS) of 11 nt or longer.

[0243] Example 9: Effect of linker length on editing yield To investigate the effect of the length of nucleotide linkers / spacers separating recruitment sequences on editing yield, several artificial nucleic acids (guide RNAs) were constructed, each containing a linker and spacers with 0, 1, 2, 3, 4, 5, and 10 adenosine nucleotides, respectively. The upper left portion of Figure 9A is a diagram used to show the interaction between gRNA and mRNA. The lines and patterns, as shown in the legend in the upper right of Figure 9A, show the substructures of gRNA (targeting sequence, spacer, first recruitment sequence, linker, second recruitment sequence, linker, third recruitment sequence) and target mRNA (target sequence, distance to next binding region, binding region #1, distance to next binding region, binding region #2, distance to next binding region, binding region #3). The recruitment sequences linked to each other via linkers, and the targeting sequences linked to the first recruitment sequence via spacers, bind to the same binding region on the mRNA for all the artificial nucleic acids compared in this example, as shown at the bottom of Figure 9A. Figure 9B shows the differences between gRNAs with different numbers of adenosine nucleotides between recruitment sequences.

[0244] Edit yield (shown as a relative multiplier change to the design using three adenosine nucleotides as linkers) is shown in Figures 9A and 9B, next to the corresponding gRNA binding site (Figure 9A) or gRNA composition (Figure 9B), respectively. 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0245] As shown in Figure 9A (gRNA binding region on mRNA) and Figure 9B (composition of guide RNA), the linker length has a relatively small effect on the editing yield in this invention. However, an optimal linker length of three adenosines between RSs was observed.

[0246] Example 10: Optimization of the arrangement of three recruitment sequences on target mRNA To evaluate the effect of the distance between regions on the target mRNA to which individual recruitment sequences bind, we constructed several artificial nucleic acids (guide RNAs) each containing three 15-nucleotide recruitment sequences that bind to specific regions on the target RNA at either short distances (15–62 nucleotides) or long distances (375–460 nucleotides). The constructs and their positions on the target mRNA are shown in Figure 10.

[0247] 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0248] As shown in Figure 10, the position of individual RSs affects the RNA editing yield, preferring to be close to the target site. However, as long as one RS is not located near the target site, other RSs can bind to fairly distant regions without significant loss of efficiency, highlighting the large sequence space available for the flexibility and optimization of guide RNA design in the present invention.

[0249] Example 11: Optimization of the placement of extended RS(20nt) in the context of two short RS(15nt) on target mRNA. To evaluate the correlation between the distance of the recruitment sequence binding region on target mRNA and the length of the recruitment sequence, several artificial nucleic acids (guide RNAs) were constructed, each having two 15nt RSs and one 20nt RS. These bind to specific regions on target RNA at either short distances (15–62 nucleotides) or long distances (375–460 nucleotides), respectively. The constructs and their positions on target mRNA are shown in Figure 11.

[0250] 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0251] As shown in Figure 11, the position of the RS very close to the target site (short) is preferable to the other settings (long). This is consistent with Example 10, Figure 10. However, the placement of the extended RS (20nt) is most effective at the 3' end of the guide RNA in both settings (long and short). Therefore, the placement of the extended recruitment sequence (RS) at the 3' end of the guide RNA may increase the editing yield regardless of the distance from RS to RS (short vs. long). Hence, the 3' terminal RS is preferably longer than the other RSs. The lengths of RS #1 and #2 do not appear to be very important.

[0252] Example 12: Effect of recruitment sequence masking on editing yield (Simulation of guide RNA with strong secondary structure in the antisense region) To evaluate the effect of recruitment sequence masking, several artificial nucleic acids (guide RNAs) were constructed, each containing three recruitment sequences (RS#1, RS#2, RS#3) that bind to specific regions on the target mRNA, and additional recruitment sequences (RS#4, RS#5, RS#6) to varying degrees, thereby simulating guide RNAs with strong secondary structures in the antisense region. The constructs are shown in Figure 12. The upper left of Figure 12A is a diagram used to show the interaction between gRNA and mRNA. The lines and patterns, as shown in the legend in the upper right of Figure 12A, show the substructures of gRNA (targeting sequence, spacer, first recruitment sequence, linker, second recruitment sequence, linker, third recruitment sequence) and target mRNA (target sequence, distance to next binding region, binding region #1, distance to next binding region, binding region #2, distance to next binding region, binding region #3). As shown in the schematic diagram of binding in Figure 12A, for all artificial nucleic acids compared in this example, the recruitment sequence and targeting sequence bind to the same binding region on the mRNA. Binding regions where the corresponding recruitment sequence in the gRNA is masked by a masking recruitment sequence (mRS) are highlighted with a vertical line pattern. The masking recruitment sequence (mRS) folds back to the actual mRNA-binding recruitment sequence, thereby preventing these recruitment sequences from interacting with their respective binding regions. Binding regions that are not masked by the masking of the recruitment sequence in the gRNA and therefore can be used for gRNA-mRNA interaction are highlighted with a right-sloping line pattern. The schematic diagram of gRNA in Figure 12A shows the differences between gRNAs. These differences are additional masking recruitment sequences (mRS) that fold back to the actual mRNA-binding recruitment sequence, thereby masking them for the purpose of gRNA-mRNA interaction.

[0253] 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0254] As shown in Figure 12A, guide RNA with masked recruitment sequences exhibits a significantly reduced editing yield compared to guide RNA containing three unmasked nucleotide recruitment sequences (3×RS gRNA, unmasked). The editing yield decreased progressively with increasing numbers of masked recruitment sequences. Therefore, this experiment suggests that guide RNA with strong secondary structures in the antisense moieties (RS and targeting sequences) exhibits a reduced editing yield and should be avoided, and thus should be included in the computer-aided design of guide RNA sequences.

[0255] Figure 12B shows the minimum free energy (MFE) secondary structure of the guide RNA construct used in Example 12. A comparison with the MFE structure in Figure 8B demonstrates how efficiently the described software enables screening for gRNAs with weak secondary structures and highlights its importance in this experiment.

[0256] Example 13: In vivo editing of dual luciferase W417X amber reporter in C57BL / 6 mice after HDTV injection of gRNA and reporter plasmid. To investigate the editing capability of the artificial nucleic acid of the present invention in animal experiments, the artificial nucleic acid (guide RNA) was delivered to C57BL / 6 mice via hydrodynamic tail vein (HDTV) injection.

[0257] C57BL / 6 mice were treated with an endotoxin-free plasmid (NucleoBond® Xtra Midi EF, Macherey Nagel) diluted in saline to a total volume equal to 10% of their body weight. This total volume was injected hydrodynamically into the tail vein (HDTV) within 5–10 seconds. Negative control mice were treated with 10 μg of dual luciferase wt / amb reporter plasmid, positive control mice with 10 μg of dual luciferase wt / wt reporter plasmid, and edited mice with 5 μg of dual luciferase wt / amb reporter plasmid and 25 μg of guide RNA plasmid. 72 hours after injection, the mice were sacrificed. Liver lobes were individually excised. Each lobe was cut into three fragments, which were then pooled together with fragments from other lobes to obtain three sample categories per mouse, each containing an equal volume of each liver lobe (Sample Categories A, B, and C). Category A samples were used for the luciferase assay, Category B samples for RNA isolation, and Category C samples were stored at -80°C as backup material. Category B and C samples were immediately frozen in liquid nitrogen. Category A samples were homogenized in 500 μl of 1× passive lysis buffer using a micropestle. After rotating the samples for 5–10 seconds, 50 μl of sample per well was transferred to a white 96-well LumiNunc plate (Thermofisher). Each sample was measured three times using 35 μl of each assay substrate per well with a Tecan Spark 10M plate reader equipped with an auto-injector. Category B samples were homogenized in 1.5 ml of Eppendorf tubes using 1 ml of TRIzol reagent (Thermofisher) and a micropestle. 200 μl of chloroform was added and vortexed for 30 seconds, then incubated at room temperature for 5–10 minutes. The mixture was then centrifuged at 4°C and 12,000 g for 20 minutes. The aqueous phase was then transferred to a new tube and 700 μl of ice-cold isopropanol was added.After precipitation overnight at -20°C, the precipitate was centrifuged at 14,000 rpm for 60 minutes and washed twice with 500 μl of 75% EtOH. After centrifugation at 14,000 rpm for 5 minutes, the pellet was dried at 50°C for 3 minutes and then dissolved in 87.5 μl of nuclease-free water. Following TRIzol isolation, the RNA was digested with DNase-I by adding 10 μl of DNase-I buffer and 2.5 μl of DNase-I (NEB) at 37°C for 30 minutes. Next, the RNA was washed twice using the RNeasy Mini RNA Isolation Kit (QIAGEN). Reverse transcription was performed using ProtoScript II reverse transcriptase (NEB), a random primer mix (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems), and 1 μg of total RNA. After PCR cleanup (NucleoSpin Gel and PCR Clean-up kit, Macherey Nagel), 2.5 μl of cDNA was used for Q5 polymerase (NEB) PCR with primer pair 2898+2899, followed by nested PCR using primer pair 3850+3851. Sanger sequencing (MWG Eurofins Genomics) was performed using primer 3850.

[0258] As shown in Figure 13, in mice treated with 5 μg of dual luciferase wt / amb reporter plasmid and 25 μg of guide RNA plasmid luciferase, activity was restored to 10.6% compared to the positive control group, as indicated by the recovery of the luciferase signal (protein level). Compared to the negative control group, the luciferase activity of the "edited group" treated with the artificial nucleic acid (guide RNA) of the present invention increased by approximately >1000-fold. The recovery of the luciferase signal (10.6% of the positive control) was confirmed at the RNA level by Sanger sequencing after RT-PCR (10.6% edit yield). Representative Sanger sequencing traces and edit yields are shown.

[0259] Example 14: Editing of Endogenous Untranslated Region (UTR) Targets Using Endogenous ADAR1 in HEK293FT Cells Ras-related protein Rab-7a is a protein encoded by the RAB7A gene in humans, and mutations in the RAB7A gene are associated with several diseases including, for example, Charcot-Marie-Tooth disease.

[0260] To evaluate editing of the UTR target sequence of the RAB7A gene, HEK293FT cells were transfected with the artificial nucleic acid (guide RNA) of the present invention.

[0261] 60,000 HEK293FT cells were seeded in 500 µl of DMEM + 10% FBS in a 24-well scale. After 24 hours, transfection was performed with 1200 ng of guide RNA plasmid (transfection grade) using FuGene6 at a 1:3 ratio. 48 hours after transfection, cells were harvested, followed by One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 µl of 10 µM sense oligo and heated to 70°C to separate residual guide RNA from the target mRNA. Then, the RT-PCR mixture was added and PCR was performed. This was followed by 1.4% agarose gel electrophoresis, PCR cleanup and Sanger sequencing (MWG).

[0262] As shown in Figure 14, the guide RNA of the present invention provides an average editing yield of 20 to 44%, along with the best editing at the RAB7A UTR target site in HEK293FT cells. In particular, there was no detectable bystander editing around the target site. By way of example, a Sanger sequencing trace is provided for the RAB7A target site (see Figure 14, lower panel). Further analysis of the RS binding region of this target is shown in Figure 16 (LEAPER benchmark).

[0263] Example 15: Editing of Several Endogenous Open Reading Frame (ORF) Targets Using Endogenous ADAR1 in HEK293FT Cells The NUP43 gene encodes nucleoporin 43, a component of the nuclear pore complex that affects bidirectional transport of macromolecules between the cytoplasm and the nucleus, and mutations in the NUP43 gene are associated with several diseases including Fanconi anemia, complementation group L, and familial atrial fibrillation.

[0264] GusB is a gene encoding β-glucuronidase involved in the degradation of glucosaminoglycans, and mutations in the GusB gene are associated with several diseases including mucopolysaccharidosis type VII and mucopolysaccharidosis type VI.

[0265] To assess the editing of ORF target sequences of the NUP43 and GusB genes, HEK293FT cells were transfected with the artificial nucleic acid (guide RNA) of the present invention.

[0266] 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, transfection was performed with 1200 ng of transfection-grade guide RNA plasmid using FuGene6 at a ratio of 1:3. Forty-eight hours after transfection, cells were harvested, followed by one-step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, samples were mixed with 1 μl of 10 μM sense oligo and heated to 70°C to separate residual guide RNA from target mRNA. The RT-PCR mixture was then added and PCR was performed. This was followed by 1.4% agarose gel electrophoresis, PCR cleanup and Sanger sequencing (MWG).

[0267] As shown in Figure 15, the guide RNA of the present invention provides an editing yield of up to 38% when targeting the ORF site L456L in GUSB in transfected HEK293FT cells, for example. In particular, bystander off-target editing is almost absent around the target site. Exemplary Sanger sequencing traces around the target sites for GUSB L456L and NUP43 V233V are shown (Figure 15, lower panel). Further analysis of the RS binding region of the NUP43 target is shown in Figure 16 (LEAPER benchmark).

[0268] Example 16: Benchmark comparing the yield on the target of the present invention and undesirable bystander out-of-target editing with prior art LEAPER gRNA. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, the cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene6 in a 1:3 ratio. 48 hours after transfection, the cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added, and PCR was performed. This was followed by 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG).

[0269] As shown in Figures 16A and 16B, the guide RNA of the present invention enables efficient editing on the target with high yield while avoiding undesirable bystander off-target editing. Data show that while the in silico selection process of RS results in a significant reduction or even complete avoidance of bystander editing, the 111nt LEAPER guide RNA lacks the design flexibility necessary to reliably avoid bystander editing. The LEAPER guide RNA was designed as an unstructured 111nt guide RNA symmetrically positioned around the target site, following Qu et al. (Nature Biotech, above). As in their previous study, the LEAPER guide RNA induced a large amount of bystander off-target editing at various sites, e.g., up to 20 sites in RAB7A, up to a maximum editing yield of 50% (see Figure 16B). Following their previous study, A:G mismatches were included to suppress bystander editing. However, in one case (NUP43), complete suppression of bystander editing was not possible (see Figure 16A). In another case (RAB7A), bystander editing could be suppressed, but at the expense of target editing (see Figure 16B). In contrast, the guide RNA designed according to the present invention enabled significantly improved editing specificity (almost no bystander off-target editing) with comparable target editing yield.

[0270] Example 17: Generation of artificial nucleic acid sequences for site-directed editing of target RNA using in silico optimization.

[0271] To facilitate the generation of a recruitment portion sequence of an artificial nucleic acid (guide RNA) containing at least one recruitment sequence, preferably a cluster of recruitment sequences that bind to first and further regions of a specific target mRNA to be edited, it is preferable to use an in silico approach to detect sequences on the target mRNA that are suitable as binding sites for the recruitment sequences and to optimize the artificial nucleic acid (guide RNA) with respect to potential intramolecular base pairing events, for example. Exemplary steps for in silico optimization of a recruitment cluster are shown in Figures 17A-17C.

[0272] Numerous presets are created by the user before the recruitment cluster in silico optimization is performed by the computer. In particular, the user-input presets are created for the following: - The sequence of the target RNA to be edited; - The length of the targeting sequence (TS) that binds to the target sequence on the target RNA (e.g., 16 to 40 nucleotides); - Length of the recruitment sequence (e.g., 11-16 nucleotides); - The distance on the target RNA between the target sequence and the sequence bound by the first recruitment sequence (e.g., 10 to 100 nucleotides); - The distance between the regions on the target RNA that are bound by the first and subsequent recruitment sequences (binding sites) (e.g., 10 to 100 nucleotides); - Length of the nucleotide linker (e.g., AAA); - The array of the second recruitment part (e.g., R / G motif): - Any 3' end sequence (e.g., UUU).

[0273] Based on the presets entered by the user, candidate guide RNAs suitable for site-specific editing of the input target RNA are created in the "Recruitment Cluster In Silico Optimization" process.

[0274] Figures 17A–17C do not illustrate the exact combination implementation used by the algorithm to process the input data, but rather explain the ideas behind the tool in a conceptual way for better understanding.

[0275] As shown in Figures 17A-17C, in step 1 of the recruitment cluster in silico optimization method, the input cDNA corresponding to the target mRNA is screened for candidate recruitment sequence binding regions containing only G, C, T, and GA (no editing is performed if the guanosine nucleotide is located 5' relative to the adenosine nucleotide in the target mRNA). For example, the screening is performed at 5' of the target sequence containing the nucleotide to be edited. In step 2, starting from the target sequence, recruitment sequence binding regions located at 5' at a specific distance (e.g., 10-100 nucleotides) from the target sequence are detected. In step 3, these recruitment sequence binding regions are selected and analyzed for their size (e.g., 11 and 13 nucleotides). In step 4, derivatives of the recruitment sequence binding region are generated using the input recruitment sequence size (e.g., 11 nucleotides). For example, if an uninterrupted recruitment sequence binding region has a length of 13 nucleotides, then when the input recruitment sequence size is set to 11 nucleotides, three derivative recruitment sequences can be created. In step 5, starting with the first set of derivative recruitment sequence binding regions (1A, 2A, 2B, 2C), the next recruitment sequence binding regions within the set range (e.g., 10 to 100 nucleotides) are detected. In step 6, the detected recruitment sequence binding regions are selected and analyzed for their size. Steps 4-6 are repeated until n (e.g., 3) recruitment sequence binding regions are selected. In step 7, the list of obtained recruitment sequence binding regions, all matching the input variables, is converted into recruitment sequences, the target sequences are converted into targeted sequences, and the generated sequences are assembled with the input R / G motif (second recruitment portion), triple adenosine linker / spacer, and three terminal uridines resulting from the U6 termination sequence.In step 8, all artificial nucleic acids (guide RNAs) in the list are folded using the Vienna RNA package (a set of standalone programs and libraries used for predicting and analyzing RNA secondary structures) to generate dot-bracket representations of these folds. The "Recruitment Cluster Finder" (RCF) allows the structures to be sorted by their free energy or by their dot-bracket ratio (ratio of dot-bracket representations). Structures with a preferred dot-bracket ratio (minimum base pairing within the antisense portion of the guide RNA) are further sorted by the lowest number of brackets in a single row within the antisense portion. The shortest one representing the weakest secondary structure receives the highest ranking. The selected recruitment sequences of the resulting guide RNAs with preferred secondary structures can then be blasted to eliminate recruitment sequences that could cause off-target editing effects within the transcriptome.

[0276] Example 18: Recruitment sequences (RS) and double-stranded ADAR recruitment domains (e.g., R / G motifs) can be positioned around a targeting sequence (TS) with certain flexibility. Luciferase assay setup for this experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well, using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system.

[0277] As shown in Figures 18A-C, the recruitment sequence can be positioned at 5' and / or 3' of the targeting sequence. While the targeting sequence typically worked very well when positioned next to the ADAR recruitment domain, other placements are possible without loss of editing efficiency.

[0278] Example 19: Correction of disease-related hIDUA W402X amber mutation in primary fibroblasts (Hurler FB, GM06214) derived from Hurler syndrome patients by transfection with chemically synthesized CLUSTER antisense oligonucleotides (ASOs).

[0279] Fibroblasts derived from patients with Scheie syndrome (GM01323) and Hurler syndrome (GM06214) were purchased from the Coriell Institute for Medical Research (USA). 2.5 × 10⁶ cells were found in 2.5 ml of DMEM + 15% FBS. 5 Cells / well were seeded in 6-well plates. For each test condition, two 6-well plates were used for the IDUA assay, and one 6-well plate was used to determine RNA editing yield by Sanger sequencing. CLUSTER ASO was a PAGE-purified, terminally blocked (2'-OMe,PS)RNA oligonucleotide with a 3×RS(20-20p8-25-20)CLUSTER design, which was ligated in-house (T4 RNA ligase) from two commercially purchased (Biospring GmbH, Germany), 69nt (5' portion) and 80nt (3' portion) HPLC-purified oligonucleotides. The complete sequence and modification patterns are shown in the list of applied gRNAs below. Transfection was performed 24 hours after seeding with 125 pmol of ASO and 7.5 μl of RNAiMAX, respectively, diluted in 250 μl of Opti-MEM. Both solutions were incubated for 5 minutes, then combined and incubated for another 20 minutes before the transfection mixture was evenly distributed into one well. The culture medium was changed 24 hours after transfection.

[0280] For Sanger sequencing: 48 hours after transfection, fibroblasts were harvested in RLT buffer (QIAGEN, #79216), followed by RNA isolation using the Monarch RNA cleanup kit (NEB, #T2030L). Turbo-DNase digestion was performed using the TURBO DNA-free Kit (Thermofisher, #AM1907). Reverse transcription was performed with random primers at 52°C for 1 hour using SuperScript IV RT (Thermofisher, #18090050). For each reaction containing 10% DMSO, conventional PCR followed by nested PCR was performed using Taq polymerase (NEB, #M267S). After the second round of PCR, the products were separated by SB-agarose gel electrophoresis. Sanger sequencing (Microsynth AG) was performed after PCR cleanup (NucleoSpin Gel and PCR Clean-up Kit, Macherey Nagel, #740609).

[0281] Regarding the α-L-idulonidase enzyme activity assay: The culture medium was changed 24 hours after transfection. 48 hours after transfection, fibroblasts were isolated and washed once with PBS. 40 μl of 0.5% Triton X-100 in PBS was added to the cell pellet, incubated on ice for 30 minutes, and the α-L-idulonidase enzyme assay was performed. For edit readout by the α-L-idulonidase enzyme activity assay, a standard dilution series of 4-methylumbelliferone (Sigma Aldrich, M1381) was prepared in 1×PBS. For each concentration, 25 μl of the standard solution was added to 25 μl of 0.4 M sodium formate buffer (pH 3.5), and 3 replicates were applied to a 96-well LumiNunc plate (VWR, 732-2696). The substrate (4-methylumbelliferyl α-L-idulonide, Glycosynth, #44076) was dissolved in 0.4 M sodium formate buffer to a final concentration of 180 μM. For the mouse IDUA assay using HeLa cells, 25 μl of 1:3 diluted cell lysate (0.5% Tween-20 / PBS) was added to 25 μl of substrate in a plate and incubated in the dark at 37°C for 45 minutes. 25 μl of undiluted cell lysate (0.5% Triton X-100 / PBS) was added to 25 μl of substrate in a plate and incubated in the dark at 37°C for 90 minutes. In both cases, the reaction was quenched by adding 200 μl of glycine carbonate buffer (0.17 M glycine / NaOH, pH 10.4). The fluorescence of 4-methylumbelliferone was measured using a Tecan Spark 10M plate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The calculated enzyme activity was based on the protein amount determined by the BCA assay (Pierce BCA Protein Assay Kit, Thermofisher, 23227). Enzyme activity was standardized to Scheie fibroblast lysates.

[0282] These experiments tested the CLUSTER approach for restoring hIDUA activity in fibroblasts isolated from Hurler patients. To overcome the strong plasmid transfection bias in these cells, CLUSTER guide RNA in the form of antisense oligonucleotides was applied. Sanger sequencing measured a guide RNA-dependent editing yield of 24% (Figure 19A), and restoration of IDUA enzyme activity to levels obtained in Scheie control fibroblasts was observed (Figure 19B). Since Scheie syndrome is a less severe disease, the data suggest that clinically beneficial effects may be within reach.

[0283] Example 20: NGS characterization of global out-of-target effects and quantification of clean editing events for CLUSTER gRNA and LEAPER gRNA.

[0284] Using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format, 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) was added 24 hours after seeding to 6 × 10⁶ cells. 4RNA editing was performed by transfection of individual HEK293FT cells. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included untargeted guide RNA (NT-RNA) and RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA. RNA was isolated using the RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated to 95°C for 3 minutes, and purified again using the RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. Libraries were prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA). RNA-seq and read mapping: A previously published pipeline was used to accurately align RNA-seq reads onto the genome (see Ramaswami, G., et al., Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods, 2012. 9(6): p. 579-81; Ramaswami, G., et al., Identifying RNA editing sites using RNA sequencing data alone. Nat Methods, 2013. 10(2): p. 128-32).Reads were aligned to the hg19 reference genome using STAR (version 2.5.3a) (see Dobin, A., et al., STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013. 29(1): p. 15-21), and clonal reads mapped to the same position (PCR duplicates) were removed by running the Picard tool (version 1.129). Of these identical reads, only those with the best mapping quality were retained for downstream analysis. Unique and non-duplicate reads were subjected to local realignment and base score recalibration using the IndelRealigner and TableRecalibration of the Genome Analysis Toolkit (GATK, version 3.6) (see Li, H., et al., The Sequence Alignment / Map format and SAMtools. Bioinformatics, 2009. 25(16): p. 2078-9). The above steps were applied separately to each RNA-seq sample. Identification of editing sites from RNA-seq data: To remove LEAPER guide RNA sequences that were incorrectly aligned to the target region, PCR duplicates in the RAB7A 3'UTR region were removed using the rmdup command in samtools (see Li, H., et al., The Sequence Alignment / Map format and SAMtools. Bioinformatics, 2009. 25(16): p. 2078-9). Furthermore, all reads containing the sequence "AAGGGTG" (3' end of LEAPER gRNA) and reads ending in "TCAAAGAC" (5' end of LEAPER gRNA) were removed. As a final step, all reads derived from the antisense sequence of the RAB7A gene were removed. This procedure was applied to all samples (CLUSTER, LEAPER, and untargeted gRNA).To call variants from mapped RNA-seq reads, we used GATK's UnifiedGenotyper (see McKenna, A., et al., The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res, 2010. 20(9): p. 1297-303). In contrast to the usual practice for variant calling, variants with relatively loose criteria were identified using the UnifiedGenotyper tool with options stand_call_conf 0, stand_emit_conf 0, and output mode EMIT_VARIANTS_ONLY. Variants derived from non-repeating and repeating non-Alu regions needed to be supported by at least three reads containing mismatches between the reference genome sequence and the RNA-seq. Support from one mismatched read was required for variants in Alu regions. This set of variant candidates was subjected to several filtering steps to improve the accuracy of editing site calling. First, all known human SNPs present in dbSNP build 137 were removed (excluding SNPs of molecular type "cDNA"; database version 135; http: / / www.ncbi.nlm.nih.gov / SNP / ), the 1000 Genomes Project, and the University of Washington Exome Sequencing Project (http: / / evs.gs.washington.edu / EVS / ).Further filtering was applied as previously described to remove false-positive RNA-seq variant calls due to technical artifacts (see Ramaswaami, G., et al., Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods, 2012. 9(6): p. 579-81; Merkle, T. and T. Stafforst, New Frontiers for Site-Directed RNA Editing: Harnessing Endogenous ADARs. Methods Mol Biol, 2021. 2181: p. 331-349). In short, the variant calling quality Q > 20 is required (see Ramaswaami, G., et al., Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods, 2012. 9(6): p. 579-81; Merkle, T. and T. Stafforst, New Frontiers for Site-Directed RNA Editing: Harnessing Endogenous ADARs. Methods Mol Biol, 2021. 2181: p. 331-349). Variants are discarded if they occur in the first 6 bases of the read, variants in simple repeats are removed (see Li, H., et al., The Sequence Alignment / Map format and SAMtools. Bioinformatics, 2009. 25(16): p. 2078-9), intron variants within 4 bp of the splice junction are removed, and variants in homopolymers are discarded. Furthermore, BLAT removed reads that mapped to highly similar regions of the transcriptome (see Kent, WJ, BLAT--the BLAST-like alignment tool. Genome Res, 2002. 12(4): p. 656-64).Finally, variants were annotated using ANNOVAR (see Wang, K., M. Li, and H. Hakonarson, ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res, 2010. 38(16): p. e164), which is based on gene models from Gencode, RefSeq, Ensembl, and UCSC. All sites identified from RNA-seq data were compared with all sites available in the RADAR database (see Ramaswami, G. and JB Li, RADAR: a rigorously annotated database of A-to-I RNA editing. Nucleic Acids Res, 2014. 42(Database issue): p. D109-13). Sites found in RADAR were called "known" sites, and those not found were called "novel" sites. Identification of significantly differently edited sites: All sites found in RNA-seq samples containing sites from the RADAR database were merged, and the editing level of edited sites with 50 read coverage (combined coverage of both repeats) was quantified. Following Fisher's exact test, Benjamini-Hochberg's multiple test correction (adjusted P<0.01) was performed to identify sites that were significantly differently edited across the entire sample (absolute editing difference >10%). Measurement of RAB7A editing precision (clean reads): To compare the specificity of CLUSTER guide RNA and LEAPER guide RNA, all mapped reads containing the edited target sequence "GCTGGCGG" were selected. AG mismatches were identified by comparing the edited reads and their partner reads with the RAB7A sequence covering the edited region. As a control, AG mismatches in reads covering the unedited target sequence "GCTAGCGG" were quantified using untargeted samples.

[0285] To compare global out-of-target effects and low levels of bystander editing between the LEAPER and CLUSTER approaches, whole-transcriptome poly(A)+ RNA sequencing experiments were performed on the RAB7A target in HEK293FT cells. Non-targeting guide RNA was transfected as a control. Using an established pipeline (see Merkle, T., et al., Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat Biotechnol, 2019. 37(2): p. 133-138), significantly differently edited sites in the transcriptome were identified, revealing a small number of hits against the LEAPER guide RNA (59) and CLUSTER (44) guide RNA, respectively (Figures 20A and 20B). This number of global out-of-target events is remarkably low compared to systems utilizing modified editases (e.g., Lambda-N-ADAR, Cas13-ADAR) where thousands of events have been reported. The NGS approach also allows us to study the frequency at which bystander editing breaks down edits on the target within the same read. 88.4% of reads using CLUSTER guide RNA were cleanly edited, compared to only 16.6% of reads using LEAPER guide RNA (Figure 20C). Considering this, the overall yield of clean edits on the target with CLUSTER guide RNA was 31%, which is clearly superior to that of LEAPER guide RNA, where only 9.2% was achieved (Figure 20D).

[0286] Example 21: A list of off-target events induced by RNA editing using CLUSTER guide RNA and LEAPER guide RNA, determined by next-generation sequencing.

[0287] RNA editing experiments and NGS data processing were performed as described in Example 20. This list shows a subset of out-of-target editing events at “unknown” sites detected by a pipeline that searches for significantly distinguishable edited sites in at least one sample when cells are treated with either CLUSTER guide RNA or LEAPER guide RNA and compared to a non-targeted control guide RNA (NT gRNA). Sites were assigned “unknown” if they were not enumerated in the RADAR database. Non-synonymous editing was detected only for a single site, HTATSF1(S742G). Mapping analysis detected sites for potential out-of-target binding of RAB7A guide RNA to CTNNAL1, HTATSF1, and ZNF740. Detected RAB7A sites represent the proportion of bystander editing sites close to the target site that meet the pipeline's significance and cutoff criteria. Data represent the mean ± sd of N=2 NGS repeats.

[0288] The number of novel, unedited sites in the non-targeted control was only 3 for CLUSTER guide RNA (in addition to the target site) and 7 for LEAPER guide RNA (Figure 21). For CLUSTER guide RNA, two of the three novel sites were exon sites (HTATSF1, CTNNAL1). Both contained putative binding sites to the guide RNA and yielded low editing yields (≤12.2%). The HTATSF1 editing site was the only one that resulted in non-synonymous amino acid changes. LEAPER guide RNA edited this site to a similar degree. The other seven novel sites detected with LEAPER guide RNA were bystander out-of-target edits very close to the target site.

[0289] [Table 6] JPEG0007914954000011.jpg255164JPEG0007914954000012.jpg255163JPEG000 7914954000013.jpg255163JPEG0007914954000014.jpg255163JPEG00079149540 00015.jpg255166JPEG0007914954000016.jpg255168JPEG0007914954000017.j pg255168JPEG0007914954000018.jpg255168JPEG0007914954000019.jpg166169

[0290] [Table 7] [Brief explanation of the drawing]

[0291] [Figure 1A] Artificial nucleic acid used in Example 1: An artificial nucleic acid molecule comprising a 20-nucleotide antisense moiety with a C / A mismatch at position 8 and a schematic R / G motif including a stem-loop structure.

[0292] B: An artificial nucleic acid molecule according to the present invention, comprising a 20-nucleotide targeting sequence (TS) having a C / A mismatch at position 8, an R / G motif having a stem-loop structure, and a cluster of three recruitment sequences (RS #1, RS #2, RS #3) having 11 to 16 nucleotides linked via an adenosine linker (AAA). [Figure 1B]Editing of a dual luciferase W417X amber reporter using endogenous ADAR1 in HeLa cells. 120,000 cells were seeded in 24-well scale. 24 hours after seeding, cells were transfected per well with 800 ng of plasmids encoding artificial nucleic acids A and B, respectively, and 200 ng of a dual luciferase reporter, using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. 72 hours after transfection, cells were harvested. RNA isolation, DNase-I digestion, and RT-PCR were followed by Sanger sequencing. [Figure 2A] Artificial nucleic acid and R / G motif version used in Example 2: (A): Prior art design having no recruitment sequence (RS) but a 16nt antisense moiety and R / G motif version 20 (RG-V20).

[0293] (B): A novel design with a recruitment cluster including three recruitment sequences (3×RS) and R / G motif version 21 (RG-V21).

[0294] (C): RG motifs version 20 and version 21 array. [Figure 2B] Editing in HeLa cells using endogenous human ADAR1 and adenovirus (AdV) encoded R / G gRNA.

[0295] 200k HeLa cells were seeded in a 96-well scale. Reverse transduction was performed at seeding using 175 MOI gRNA AdV. Forward transduction was achieved 24 hours after seeding using 175 MOI dual luciferase wt / amb AdV or 5 MOI wt / wt. Note: Firefly is always normalized on Renilla, so the amount of wt / wt AdV does not affect the final percentage. Luciferase assays were performed 96 hours after reverse infection. [Figure 3] Editing of dual luciferase reporters via recruitment of endogenous human ADAR1 using AdV-encoding 3× RS guide RNA in several cell lines. Analysis via dual luciferase assay (N=3, each with 2 technical replicates).

[0296] 25,000 cells of each cell type were seeded in a 96-well scale. In HeLa cells, forward transduction was achieved 24 hours after seeding. In all other cell types, reverse transduction was achieved at seeding. Collection and luciferase assays were performed 96 hours after infection.

[0297] Luciferase assay: 420 datapoints (35 settings, 6 Renira and 6 Fireflies per setting) Settings: gRNA MOI: HeLa(100 MOI), SK-N-BE(100 MOI), Huh7(75 MOI), A549(75 MOI), HepG2(75 MOI), SY5Y(100 MOI), U87MG(100 MOI), U2OS(75 MOI);DL wt / amb 50 MOI;DL wt / wt 5 M.O.I. [Figure 4]Editing of dual luciferase reporters via recruitment of endogenous human ADAR1 using AdV-encoding 3× RS guide RNA in several cell lines. Analysis via Sanger sequencing.

[0298] For Huh7 cells, SK-N-BE cells, A549 cells, HepG2 cells, and SY5Y cells: 25,000 cells per well (96-well scale) were reverse-infected at the MOI indicated on day 1 seeding. The culture medium was changed on days 2-4. On day 5, 6 wells were collected and pooled per RT-PCR cycle. For HeLa cells: 25,000 cells per well (96-well scale) were seeded on day 1. On day 2, forward infection was performed using the following MOIs: HeLa: n=2 [75 MOI], n=2 [100 MOI], n=2 [125 MOI]; Huh7: n=3 [75 MOI]; A549: n=3 [75 MOI]; HepG2: n=3 [75 MOI]; SK-N-BE(2): n=3 [100 MOI]; SH-SY5Y: n=3 [100 MOI]. The culture medium was changed on days 3-5. On day 6, 6 wells were collected and pooled per RT-PCR cycle. [Figure 5A] Editing of several disease-associated target mRNAs in HeLa cells using 6-9× RS guide RNA and endogenous ADAR1. 120,000 cells were seeded in 24-well scale. 24 hours after seeding, cells were transfected with 800 ng of guide RNA plasmid and 200 ng of target coding plasmid (cDNA) per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. 72 hours after transfection, cells were harvested. RNA isolation, DNase-I digestion, and RT-PCR were followed by Sanger sequencing. [Figure 5B] Figure 5A shows an exemplary Sanger sequence trace of the data, demonstrating the complete absence of off-target bystander editing around the target site. [Figure 6]The plasmid-encoding RG-V21_20p8_3×RS and RG-V21_20p8_8×RS guide RNAs primarily recruit ADAR1 isoform p110 in HeLa cells. 1.2×10⁵ HeLa cells were seeded in a 12-well scale, and reverse transfection was achieved with scrambled siRNA, ADAR1 siRNA, or ADAR1p150 siRNA using 2.5 pmol siRNA per well (3 μl HighPerfect + 2.5 μl 1 μM siRNA, and 200 μl using OptiMem). Each siRNA was seeded in 6 wells. Each well used in the 12-well plate contained the indicated 200 μl transfection mixture for the corresponding siRNA. 24 hours after siRNA transfection, similarly treated wells were harvested and the cells were pooled. Subsequently, 25,000 differently treated HeLa cells were seeded in a 96-well scale. Twenty-four hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. Luciferase assays were performed 48 hours after transfection using the Promega dual luciferase reporter assay system. [Figure 7] Effect of recruitment sequence number on editing yield. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. The location of target sequences on the luciferase transcript is shown in dark gray, and the location of each RS binding region is shown in light gray. Editing target

[0299] [ka]

[0300] The positions are also shown. Each RS is 9-16 nt long and is separated by an AAA linker. [Figure 8A] Effect of recruitment sequence length (7-15 nt / RS) on editing yield. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. [Figure 8B] Schematic diagram of the minimum free energy structure of guide RNA at 37°C. [Figure 9A] Effect of adenosine linker length on editing yield. Luciferase activity (multiplier change) is shown next to a schematic diagram indicating the corresponding gRNA-binding region (BR) on mRNA. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. [Figure 9B]The dataset in Figure 9A is shown next to a schematic diagram illustrating the corresponding gRNA composition, with respect to the number of adenosine nucleotides used as linkers between recruitment sequences (RSs) and the number of adenosine nucleotides used as spacers between recruitment sequence #1 and the target sequence. [Figure 10] Optimization of the arrangement of three recruitment sequences on target mRNA. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. (S = short distance between two RSs (15-62 nt); L = long distance between two RSs (375-460 nt)) [Figure 11] Optimization of the placement of an extended recruitment sequence (20nt) in the context of two short recruitment sequences (15nt) on target mRNA. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid vs. Lipofectamine-3000 ratio of 1:1.5. Luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. (S = short distance between two RSs (15-62nt); L = long distance between two RSs (375-460nt); RSs extended from 15nt to 20nt are highlighted) [Figure 12A]Effect of recruitment sequence masking (simulation of guide RNA with strong secondary structure in the antisense portion). The upper left portion of Figure 12A depicts the representation used to show gRNA-mRNA interaction. The binding schematic portion shows the binding region (BR) of the gRNA recruitment sequence on the mRNA. RS#1 binds to BR#1. The gRNA schematic shows the gRNA composition consisting of the R / G motif V21, targeting sequence, adenosine nucleotide linker / spacer, recruitment sequences (RS)#1-#3, and in some cases, masking recruitment sequences (mRS)#4-#6. Luciferase assay setup for characterization experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid-to-Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. [Figure 12B] Schematic diagram of the minimum free energy structure of guide RNA at 37°C. [Figure 13]Editing of the dual luciferase W417X amber reporter in C57BL / 6 mice after hydrodynamic tail vein (HDTV) injection of gRNA and reporter plasmid. Negative control mice were treated with 10 μg of dual luciferase wt / amb reporter plasmid, positive control mice were treated with 10 μg of dual luciferase wt / wt reporter plasmid, and edited mice were treated with 5 μg of dual luciferase wt / amb reporter plasmid and 25 μg of guide RNA plasmid. The guide RNA was a 20-15-15-20p8 guide RNA, and therefore contained a typical 20nt targeting sequence with three RSs (having lengths of 20nt, 15nt, and 15nt) and a cytosine mismatched with adenosine at position 8. The guide RNA further contained a V21R / G motif at its 5' end (not shown). The edits were analyzed by dual luciferase assay (protein level) and by Sanger sequencing after reverse transcription (RNA level). Exemplary Sanger sequence traces are shown. [Figure 14] Editing of endogenous UTR targets using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene 6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added and PCR was performed. This was followed by 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG). Exemplary Sanger sequencing traces are shown, indicating the editing sites and yields. "A" and "G" represent the adenosine peak and the guanosine peak, respectively. [Figure 15]Editing of endogenous ORF targets using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene 6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added and PCR was performed. This was followed by 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG). Exemplary Sanger sequencing traces are shown, indicating the editing sites and editing yields. "A" and "G" represent the adenosine peak and the guanosine peak, respectively. [Figure 16A] This study benchmarks the guide RNA of the present invention against a conventional LEAPER gRNA targeting NUP43 V233V. It also describes the editing of an endogenous target using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene 6 in a 1:3 ratio. 48 hours after transfection, cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added, and PCR was performed. This was followed by 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG). A biological replication study with N=3. [Figure 16B]This study benchmarks the guide RNA of the present invention against a conventional LEAPER gRNA targeting RAB7A 3'UTR TAG#1. It also describes the editing of an endogenous target using endogenous ADAR1 in HEK293FT cells. 60,000 HEK293FT cells were seeded in 500 μl of DMEM + 10% FBS in a 24-well scale. After 24 hours, the cells were transfected with 1200 ng of guide RNA plasmid (transfection grade) using FuGene6 in a 1:3 ratio. 48 hours after transfection, the cells were harvested and then subjected to One-Step RT-PCR using the Biotechrabbit Kit. Immediately before RT-PCR, the sample was mixed with 1 μl of 10 μM sense oligonucleotide and heated to 70°C to separate the remaining guide RNA from the target mRNA. The RT-PCR mixture was then added, and PCR was performed. Following this, 1.4% agarose gel, PCR cleanup, and Sanger sequencing (MWG) were performed. N=3 biological replicates. [Figure 17A] Recruitment cluster in silico optimization using the Recruitment Cluster Finder tool. [Figure 17B] Recruitment cluster in silico optimization using the Recruitment Cluster Finder tool. [Figure 17C] Recruitment cluster in silico optimization using the Recruitment Cluster Finder tool. [Figure 18A] Legend for Figures 18B and 18C. RM = Recruitment portion. [Figure 18B] The recruitment sequence (RS) and double-stranded ADAR recruitment domain (e.g., R / G motif) can be positioned around the targeting sequence (TS) with a certain degree of flexibility. The components of the antisense portion of the CLUSTER guide RNA targeting a dual luciferase reporter in HeLa cells were newly positioned, starting from the conventional #6-#5-#4-TS design. [Figure 18C]Results of the dual luciferase assay. Data are shown as mean ± sd of N=5 biological replicates. In particular, several designs (e.g., #4-TS-#3-#2) gave better editing yields compared to the conventional #6-#5-#4-TS design. Importantly, the former design allows for the inclusion of the sequence space 3' of targeted adenosine for binding to the CLUSTER guide RNA (e.g., binding sites #3, #2, #1). Luciferase assay setup for this experiment: 25,000 HeLa cells were seeded in a 96-well scale. 24 hours after seeding, cells were transfected with 160 ng of guide RNA plasmid and 40 ng of dual luciferase reporter per well using a plasmid vs. Lipofectamine-3000 ratio of 1:1.5. The luciferase assay was performed 48 hours after transfection using the Promega dual luciferase reporter assay system. [Figure 19A]Correction of disease-related hIDUA W402X amber mutations in primary fibroblasts (Hurler FB, GM06214) derived from Hurler syndrome patients by transfection with chemically synthesized CLUSTER antisense oligonucleotides (ASOs). Edit yield was determined by Sanger sequencing. Fibroblasts from patients with Hurler syndrome (GM06214) were purchased from the Coriell Institute for Medical Research (USA). 2.5 × 10⁵ cells / well were seeded in 2.5 ml of DMEM + 15% FBS into 6-well plates. RNA editing rate was determined by Sanger sequencing using one 6-well plate for each test condition. CLUSTER ASO is a PAGE-purified, terminally blocked (2'-OMe,PS)RNA oligonucleotide with a 3×RS(20-20p8-25-20)CLUSTER design, which was ligated in-house (T4 RNA ligase) according to our recently published protocol from two commercially purchased (Biospring GmbH, Germany), 69nt (5' portion) and 80nt (3' portion) length HPLC-purified oligonucleotides. The complete sequence and modification patterns are shown in the following list of applied gRNAs. Transfection was performed 24 hours after seeding with 125 pmol of ASO and 7.5 μl of RNAiMAX, each diluted in 250 μl of Opti-MEM. Both solutions were incubated for 5 minutes, then combined and incubated for a further 20 minutes before the transfection mixture was evenly distributed into one well. The medium was changed 24 hours after transfection. 48 hours after transfection, fibroblasts were collected in RLT buffer (QIAGEN, #79216), followed by RNA isolation using the Monarch RNA Cleanup Kit (NEB, #T2030L). Turbo-DNase digestion was performed using the TURBO DNA-free Kit (Thermofisher, #AM1907).Reverse transcription was performed using SuperScript IV RT (Thermofisher, #18090050) at 52°C for 1 hour with random primers. Using Taq polymerase (NEB, #M267S), standard PCR followed by nested PCR was performed in each reaction containing 10% DMSO. After the second PCR, the products were separated by SB-agarose gel electrophoresis. After PCR cleanup (NucleoSpin Gel and PCR Clean-up Kit, Macherey Nagel, #740609), Sanger sequencing (Microsynth AG) was performed. [Figure 19B]Furthermore, the restoration of protein function was determined using an α-L-iduronidase enzyme activity assay. The measured hIDUA enzyme activity in Hurler fibroblasts was normalized to the IDUA enzyme activity measured in Scheie patient fibroblasts (Scheie FB, GM01323). Data are presented as mean ± sd of N=4–5 biological replicates, as indicated by individual data points. Patient-derived fibroblasts from Scheie syndrome (GM01323) and Hurler syndrome (GM06214) were purchased from the Coriel Institute for Medical Research (USA). 2.5 × 10⁵ cells / well were seeded in 2.5 ml of DMEM + 15% FBS into 6-well plates. For each test condition, two 6-well plates were used for the IDUA assay. CLUSTER ASO is a PAGE-purified, terminally blocked (2'-OMe,PS)RNA oligonucleotide with a 3×RS(20-20p8-25-20)CLUSTER design, which was ligated in-house (T4 RNA ligase) according to our recently published protocol from two commercially available products (Biospring GmbH, Germany) as well as HPLC-purified oligonucleotides of 69nt (5' portion) and 80nt (3' portion) lengths. The complete sequences and modification patterns are shown in the following list of applied gRNAs. Transfection was performed 24 hours after seeding in 125 pmol of ASO and 7.5 μl of RNAiMAX, each diluted in 250 μl of Opti-MEM. Both solutions were incubated for 5 minutes, then combined and incubated for a further 20 minutes before the transfection mixture was evenly distributed into one well. The medium was changed 24 hours after transfection. 48 hours after transfection, fibroblasts were isolated and washed once with PBS. 40 μl of 0.5% Triton X-100 in PBS was added to the cell pellet, incubated on ice for 30 minutes, and an α-L-iduronidase enzyme assay was performed. A standard dilution system of 4-methylumbelliferone (Sigma Aldrich, M1381) was prepared in 1×PBS for editing readout by the α-L-iduronidase enzyme activity assay.For each concentration, 25 μl of the standard solution was added to 25 μl of 0.4 M sodium formate buffer (pH 3.5), and 3 replicates were applied to a 96-well LumiNunc plate (VWR, 732-2696). The substrate (4-methylumbelliferyl α-L-idulonide, glycosine, #44076) was dissolved in 0.4 M sodium formate buffer to a final concentration of 180 μM. For the mouse IDUA assay using HeLa cells, 25 μl of 1:3 diluted cell lysate (0.5% Tween-20 / PBS) was added to 25 μl of substrate in the plate, and incubated in the dark at 37°C for 45 minutes. 25 μl of undiluted cell lysate (0.5% Triton X-100 / PBS) was added to 25 μl of substrate in the plate, and incubated in the dark at 37°C for 90 minutes. The reaction was quenched in both cases by adding 200 μl of glycine carbonate buffer (0.17 M glycine / NaOH, pH 10.4). Fluorescence of 4-methylumbelliferone was measured using a Tecan Spark 10M plate reader at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Calculated enzyme activity was based on the protein amount determined by a BCA assay (Pierce BCA Protein Assay Kit, Thermofisher, 23227). Enzyme activity was normalized to Scheie fibroblast lysates. [Figure 20A]Analysis of off-target editing in the poly(A)+ transcriptome to recruit endogenous ADARs from 293 FT cells to the 5'UAG site in the 3'-UTR of endogenous RAB7A using CLUSTER guide RNA (19-11-13-20p8). Scatter plots show differential editing at approximately 30,000 sites, comparing editing levels in cells transfected with plasmids containing CLUSTER or non-targeting guide RNA. Experiments were performed using two independent replicates. Edits on targets are indicated by arrows. Significantly different editing sites (adjusted P<0.01, Fisher's exact test, two-tailed, N≧50) are highlighted in black. RNA editing was performed by transfecting 6 × 10⁴ HEK293FT cells with 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) 24 hours after seeding using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included non-targeted guide RNA (NT-RNA) and RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA. RNA was isolated using the RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with RNA strands that were inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again using the RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. Libraries were prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq6000 (50M reads, 2×100bp paired-end, Illumina, USA). [Figure 20B]Analysis of off-target editing in the poly(A)+ transcriptome to recruit endogenous ADAR to the 5'UAG site in the 3'-UTR of endogenous RAB7A with 111nt LEAPER guide RNA from 293 FT cells. Scatter plots show differential editing at approximately 30,000 sites, comparing editing levels in cells transfected with plasmids containing LEAPER or non-targeting guide RNA. Experiments were performed using two independent replicates. Edits on targets are indicated by arrows. Significantly different editing sites (adjusted P<0.01, exact test, two-tailed, N≧50) are highlighted in black. RNA editing was performed by transfecting 6 × 10⁴ HEK293FT cells with 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) 24 hours after seeding using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included untargeted guide RNA (NT-RNA) and RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated with RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with an RNA strand that was inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and re-purified with RNeasy MinElute Kit. Purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. Libraries were prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq6000 (50M reads, 2×100bp paired-end, Illumina, USA). [Figure 20C]Edit accuracy was evaluated by analyzing all NGS reads with targeted edits for bystander editing. CLUSTER guide RNA primarily yielded clean sequencing reads and a small percentage of reads with a single bystander edit, while reads from LEAPER samples very frequently contained several bystander edits. RNA editing was performed by transfecting 6 × 10⁴ HEK293FT cells with 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) 24 hours after seeding using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These configurations included (1) non-targeting guide RNA (NT-RNA), (2) RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA, and (3) RAB7A 3'UTR 111p56 LEAPER guide RNA. The RNAs were isolated using the RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with RNA strands that were inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again using the RNeasy MinElute Kit. The purified RNAs were delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. The library was prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA). [Figure 20D]Estimation of yield for clean editing (without bystander) and all edits on the target. RNA editing was performed by transfecting 6 × 10⁴ HEK293FT cells with 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) 24 hours after seeding using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included (1) untargeted guide RNA (NT-RNA), (2) RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA, and (3) RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated using the RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with RNA strands that were inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and re-purified using the RNeasy MinElute Kit. The purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. Libraries were prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA). [Figure 21]A list of off-target events induced by RNA editing using CLUSTER guide RNA and LEAPER guide RNA, determined by next-generation sequencing. This list represents a subset of off-target editing events at "unknown" sites detected by a pipeline searching for significantly distinguishable edited sites in at least one sample when cells are treated with either CLUSTER guide RNA or LEAPER guide RNA and compared to a non-targeting control guide RNA (NT gRNA). A site was assigned "unknown" if it was not enumerated in the RADAR database. Non-synonymous editing was detected only for a single site, HTATSF1(S742G). Mapping analysis detected sites for potential off-target binding of RAB7A guide RNA to CTNNAL1, HTATSF1, and ZNF740. Detected RAB7A sites represent the proportion of bystander editing sites close to the target site that meet the pipeline's significance and cutoff criteria. Data represent the mean ± sd of N=2 NGS repeats. RNA editing was performed by transfecting 6 × 10⁴ HEK293FT cells with 1200 ng of guide RNA plasmid (NucleoSpin Plasmid Transfection-grade, Macherey Nagel, #740490) 24 hours after seeding using FuGene6 (Promega, #E2691) in a 1:3 ratio in a 24-cell format. Cells were harvested 48 hours after transfection. In total, three settings were performed, each with independent replicates. These settings included (1) untargeted guide RNA (NT-RNA), (2) RAB7A 3'UTR 19-11-13-20p8 CLUSTER guide RNA, and (3) RAB7A 3'UTR 111p56 LEAPER guide RNA. RNA was isolated using the RNeasy MinElute Kit (Qiagen, #74204), treated with DNase I (NEB, #M0303S), incubated with RNA strands that were inversely complementary to the antisense portion of each guide RNA, heated at 95°C for 3 minutes, and purified again using the RNeasy MinElute Kit.Purified RNA was delivered to CeGaT (Germany) for poly(A)+mRNA sequencing. Libraries were prepared from 200 ng of RNA using the TruSeq Stranded mRNA Library Prep Kit (Illumina, USA) and sequenced using NovaSeq 6000 (50M reads, 2×100bp paired-end, Illumina, USA).

Claims

1. An artificial nucleic acid for site-directed editing of a target RNA, comprising the following in the direction from 5' to 3' or from 3' to 5': a) A first recruitment portion capable of recruiting deaminase, the first recruitment portion comprising a cluster of recruitment sequences, each comprising three recruitment sequences and a nucleotide linker linking the recruitment sequences of the first recruitment portion, wherein the recruitment sequences bind to first, second, and third recruitment sequence binding regions in the target RNA, each of the three recruitment sequences comprising at least 10 nucleotides, and each nucleotide linker comprising 2 to 6 adenosine nucleotides; b) A target sequence in the target RNA containing one or more nucleotides to be edited that is complementary to or A targeting sequence comprising at least 10 nucleotides, which includes nucleic acid sequences that are at least partially complementary, and c) A second recruitment portion capable of recruiting deaminase, Here, the artificial nucleic acid includes a nucleotide spacer between the first recruitment sequence and the targeting sequence of the artificial nucleic acid, and the nucleotide spacer contains 2 to 6 adenosine nucleotides. Artificial nucleic acid.

2. The artificial nucleic acid according to claim 1, wherein each recruitment sequence includes a nucleic acid sequence that is complementary or at least partially complementary to the binding region of each recruitment sequence in the target RNA.

3. The artificial nucleic acid according to claim 1, wherein each of the three recruitment sequences contains 10 to 200 nucleotides.

4. The artificial nucleic acid according to any one of claims 1 to 3, wherein the first recruitment sequence binding region in the target RNA, and / or the second and / or further recruitment sequence binding regions in the target RNA, do not contain an editable adenosine nucleotide.

5. The aforementioned spacer, (i) A first recruitment sequence binding region in the target RNA, which is bound by a first recruitment sequence positioned adjacent to the targeting sequence of the artificial nucleic acid, and (ii) The target sequence in the target RNA An artificial nucleic acid according to any one of claims 1 to 4, which does not bind to or is not complementary to any of the above.

6. The artificial nucleic acid according to any one of claims 1 to 5, wherein the targeting sequence includes a cytidine nucleotide that mismatches with the adenosine to be edited at a position corresponding to the nucleotide to be edited.

7. The artificial nucleic acid according to claim 6, wherein the cytidine nucleotide that mismatches the adenosine to be edited is located at least six nucleotides away from either the 5' or 3' end of the targeted sequence.

8. The artificial nucleic acid according to any one of claims 1 to 7, wherein the second recruitment portion includes a nucleic acid sequence that can bind to a deaminase without binding to the target RNA.

9. The artificial nucleic acid according to claim 8, wherein the nucleic acid sequence can bind to the dsRNA-binding domain of a deaminase.

10. The artificial nucleic acid according to claim 8 or 9, wherein the deaminase is adenosine deaminase.

11. The artificial nucleic acid according to claim 9, wherein the nucleic acid sequence can bind to ADAR1 or ADAR2.

12. The artificial nucleic acid according to any one of claims 9, 10, or 11, wherein the nucleic acid sequence is capable of forming intramolecular base pairs.

13. The artificial nucleic acid according to claim 12, wherein the nucleic acid sequence is capable of forming a stem-loop structure comprising a double helix stem containing at least two mismatches and a loop consisting of 3 to 8 nucleotides.

14. The artificial nucleic acid according to claim 13, wherein the nucleic acid sequence comprises a nucleotide sequence of 5'-GGUGU CGAGA AGAGG AGAAC AAUAU GCUAA AUGUU GUUCU CGUCU CCUCG ACACC-3' or a nucleotide sequence of 5'-GUG GAA UAG UAU AAC AAU AUG CUA AAU GUU GUU AUA GUA UCC CAC-3'.

15. A composition comprising the artificial nucleic acid described in any one of claims 1 to 14.

16. An artificial nucleic acid according to any one of claims 1 to 14, or a composition according to claim 15, for use as a pharmaceutical.

17. An artificial nucleic acid according to claim 16, or a composition according to claim 16, for use in the treatment or prevention of a genetic disease or genetic disorder.

18. The hereditary disease or hereditary disorder is selected from the group consisting of metabolic diseases, neoplastic diseases, autoimmune diseases, cardiovascular diseases and neurological diseases, as described in claim 17, the artificial nucleic acid or composition described in claim 17.

19. (i) A genetic disorder or genetic disorder selected from the group consisting of metabolic disorders, neoplastic disorders, autoimmune disorders, cardiovascular disorders and neurological disorders, or (ii) A disease or disorder selected from the group consisting of infectious diseases, neoplastic diseases, cardiovascular diseases, autoimmune diseases, allergies, and neurological diseases or disorders. An artificial nucleic acid according to any one of claims 1 to 14, or the composition according to claim 15, for use in the diagnosis of [condition].

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