ADAR guide RNA and use thereof

Abstract

The present application relates to an ADAR guide RNA (agRNA) for RNA editing. Specifically, the agRNA contains: (1) a targeting region complementarily paired with a target RNA; and (2) a recruitment region complementarily paired with a target RNA and having no stem-loop structure, wherein the recruitment region is capable of binding to and / or recruiting an editing entity. Further provided in the present application are a composition containing the agRNA, and the use of the agRNA and the composition.

Description

ADAR guide RNA and applications thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an ADAR guide RNA (agRNA), a composition and application thereof. BACKGROUND

[0002] Gene editing technology has become one of the leading technologies in the field of life sciences in recent years, and has shown great potential in disease treatment. Nucleic acid drugs developed based on gene editing technology have unique advantages compared to traditional small molecule drugs or macromolecular antibody drugs. They can directly repair DNA or RNA to intervene from the etiological level to achieve therapeutic effect, especially in the development of therapies for genetic diseases, chronic diseases and tumors. Gene editing technology is expected to bring substantial medical help to countless patients.

[0003] Gene editing technology can be divided into DNA editing and RNA editing according to the target. Compared with DNA editing, RNA editing repairs mutations or regulation at the RNA level without changing the original sequence of the genome, which makes RNA editing reversible and safer, thus having significant advantages in certain disease scenarios. RNA editing mainly involves single-base editing technology, the core principle of which is to change specific nucleotide bases through the deamination process of adenosine deaminase to repair gene function and achieve the purpose of disease treatment.

[0004] Specifically, targeted RNA editing technology currently mainly includes two types of paths. The first type is a two-molecule editing system, which contains an exogenous deaminase and a guide RNA. The exogenous deaminase realizes the editing of the target base by accurately recognizing and deaminating the specific RNA sequence. The guide RNA is responsible for guiding the deaminase to the specific RNA target sequence to ensure the efficiency and accuracy of the editing process. The second type is mainly a single-molecule system, which only needs to design a specific guide RNA and deliver it to the cells of the target organ to recruit the endogenous deaminase ADAR protein expressed in the cells and edit the specific site. The overexpression of deaminase caused by the two-molecule system may lead to the risk of cell carcinogenesis, so its clinical application has great limitations. In the single-molecule system, the guide RNA plays two roles; one is to target the site that needs to be edited, and the other is to form a specific structure within the editing region to recruit ADAR protein. Therefore, the existing guide RNA design based on a single molecule mainly has two types; one is a single-stranded guide RNA that is complementary to the target RNA for editing and simultaneously serves as a recruitment enzyme and a targeting function, and the other is a part of the sequence that is complementary to the target RNA for targeting and another part of the sequence that forms an intramolecular double-stranded structure to recruit ADAR protein for recruitment.

[0005] RNAs in cells naturally fold into a variety of different multi-dimensional structures, which makes it difficult to design guide RNAs for good editing efficiency according to the prior art. The present application discloses a new guide RNA design strategy, which is a non-continuous guide agRNA that binds to a target RNA, the guide agRNA comprises two domain structures, the first domain is a targeting region, and the second domain is a recruiting region, wherein the recruiting region is an intermolecular RNA double-stranded structure formed by the guide agRNA and the target RNA, and the two domains are connected by a linker. SUMMARY

[0006] The present application provides an ADAR guide RNA (agRNA) for RNA editing, the agRNA comprises: (1) a targeting region that is complementary to a target RNA; and (2) a recruiting region that is complementary to the target RNA and does not have a stem loop structure, the recruiting region can bind and / or recruit an editing entity. The present application provides a new RNA editing strategy, which requires the use of the agRNA described in the present application. Compared with separately transfecting a targeting region sequence, separately transfecting a recruiting region sequence, mixed transfecting two region sequences, and transfecting a traditional guide RNA with an intramolecular recruiting region, the agRNA provided in the present application has higher editing efficiency in different genes; and the agRNA with different lengths of the targeting region and the recruiting region connected by a linker, the agRNA with different distances between the fixed recruiting region position and the recruiting region, and the agRNA with different linkers, compared with the control group, the agRNA provided in the present application has higher editing efficiency.

[0007] In one aspect, the agRNA provided in the present application comprises a targeting region and a recruiting region, the targeting region is complementary to a part of the target RNA containing a target site, and the recruiting region is not complementary to the part of the target RNA containing the target site.

[0008] In certain embodiments, the length of the targeting region is greater than 6 nt. In certain embodiments, the length of the targeting region is 10-15 nt.

[0009] In certain embodiments, the length of the recruiting region is greater than 10 nt. In certain embodiments, the length of the recruiting region is 20-25 nt.

[0010] In certain embodiments, the editing entity comprises deaminase activity. In certain embodiments, the editing entity comprises an adenosine deaminase (ADAR) that acts on RNA. In certain embodiments, the editing entity comprises hADAR1 and hADAR2.

[0011] In certain embodiments, the target site is adenosine.

[0012] In certain embodiments, the targeting region and the recruiting region are connected by a linker.

[0013] In certain embodiments, the linker is optionally selected from the group consisting of: one or more unmodified nucleotides, one or more modified nucleotides, oligopeptides, and any other chemical linker. In certain embodiments, the linker is optionally selected from the group consisting of: alkyl, PEG chain, annealing into double-stranded RNA, and RNA base sequence. In certain embodiments, the linker is PEG2, PEG6, C3, C6, or C9.

[0014] In certain embodiments, the agRNA comprises a chemical modification. In certain embodiments, the chemical modification comprises one or more of base modification, sugar modification, backbone modification, and combinations thereof. In certain embodiments, the chemical modification is optionally selected from the group consisting of: LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, base substitution modification (thymine, 5-methylcytosine, and inosine), and phosphorothioation modification.

[0015] In certain embodiments, the agRNA is perfectly complementary to the target RNA.

[0016] In certain embodiments, the agRNA is complementary to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges.

[0017] In certain embodiments, the target RNA is optionally selected from the group consisting of: pre-mRNA, mRNA, rRNA, tRNA, Inc-RNA, snRNA, snoRNA, and microRNA.

[0018] In another aspect, the present application provides a delivery vehicle comprising the agRNA described herein.

[0019] In another aspect, the present application provides a pharmaceutical composition comprising the agRNA described herein, and / or the delivery vehicle described herein.

[0020] In another aspect, the present application provides a plasmid for expressing the full-length and / or partial sequence of the agRNA described herein.

[0021] In another aspect, the present application provides a method for site-directed editing of a nucleotide in a target RNA in a eukaryotic cell, comprising using the agRNA described herein.

[0022] In another aspect, the present application provides the agRNA, the delivery vector, the pharmaceutical composition for use in the manufacture of a medicament for preventing and / or treating a disease and / or a disorder.

[0023] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the application. As will be realized, the application is capable of modifications in various obvious aspects, all without departing from the application as described and claimed. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0024] The features and advantages of the application involved will be better understood from the following detailed description of exemplary embodiments with reference to the drawings. A brief description of the drawings is as follows:

[0025] FIG. 1 shows the results of different gene tests according to the present application, indicating that agRNA connected by a linker can significantly improve editing efficiency compared to mixed targeting regions and recruitment regions, ACTB gene (FIG. 1A, 1B) and GAPDH gene (FIG. 1C, 1D) corresponding to the horizontal coordinates, respectively, are the sequence of the targeting region transfected alone, the sequence of the recruitment region transfected alone, the mixed transfection of the two regions and the sequence after the linker connection, OGT gene (FIG. 1E, 1F) adds the design of the recruitment domain with secondary structure on this basis.

[0026] FIG. 2A shows the improvement of target gene editing efficiency by connecting different lengths of targeting regions and recruitment regions by a linker according to the present application, FIG. 2A is the test result in Hela cells, FIG. 2B is the test result in HEK293 cells, the horizontal coordinates correspond to the sequence of the targeting region transfected alone, the sequence of the recruitment region transfected alone, the mixed transfection of the two regions and the design of the present application, respectively.

[0027] FIG. 3 shows that the editing efficiency of two region sequences connected by different linkers according to the present application is significantly higher than that of mixed transfection sequences without a linker, wherein FIG. 3A is the editing efficiency in Hela cells, and FIG. 3B is the editing efficiency in HEK293 cells.

[0028] FIG. 4 shows the editing effect of the fixed recruitment region position test distance from the recruitment region according to the present application, wherein FIG. 4A and 4B have a 9bp interval at site2, FIG. 4C and 4D have a 5bp interval at site3, and FIG. 4E and 4F have a 379bp interval at site4. Higher editing can be achieved under these different intervals.

[0029] Figure 5 shows that the agRNA described in the present application can efficiently edit the reporter gene with a specific structure, the reporter gene is designed to have a 17 bp double-stranded structure between the target region and the recruitment region bound by the agRNA to simulate the target with such secondary structure, and the results show that the linker can significantly improve the editing efficiency, Figure 5A is the result of Hela cells, and 5B is the result of HEK293 cells.

[0030] Figure 6 shows the difference in editing level of the reporter gene by different design strategies described in the present application, the abscissa respectively represents co-transfection of reporter gene plasmid plus traditional continuous single-stranded guide RNA, co-transfection of reporter gene plasmid plus traditional guide RNA with intramolecular recruitment region and co-transfection of reporter gene plasmid plus agRNA described in the present application, Figure 6A is the editing effect in Hela cells, and Figure 6B is the editing effect in HEK293 cells. DETAILED DESCRIPTION

[0031] The following specific embodiments illustrate the embodiments of the present application, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the present application.

[0032] Term definition

[0033] In the present application, the term "RNA editing" generally refers to RNA regulation by editing the target RNA. In the present application, the purpose of RNA editing can be to change the base of the target RNA, and can be to regulate the gene expression amount of the target RNA. The editing of adenosine to inosine in the target RNA can be achieved by expressing a specially designed RNA to recruit endogenous deaminase ADAR in the cell.

[0034] In the present application, the term "antisense oligonucleotide", also known as "Antisense Oligonucleotide", abbreviated as "ASO", generally refers to an artificially synthesized single-stranded or double-stranded oligonucleotide, which can be modified by modifying the structure, binding site, etc. In the present application, the ASO can be in single-stranded form, and its function can be to complementarily pair with a specific sequence of the target RNA and edit it.

[0035] In the present application, the term "antisense oligonucleotide guide RNA", also known as "Antisense Oligonucleotide guide RNA", abbreviated as "agRNA", generally refers to an artificially synthesized single-stranded or double-stranded oligonucleotide, which can be modified by modifying the structure, binding site, etc. In the present application, the agRNA can be in single-stranded form, and its function can be to complementarily pair with a specific sequence of the target RNA and edit it.

[0036] In the present application, the term “editing entity”, also referred to as “RNA editing entity”, can be used interchangeably in the present application, and generally refers to a molecule directly involved in and leading to the change of a molecule, which can be an enzyme or a small molecule RNA. In the present application, the editing entity can have deaminase activity, which can be hADAR1 and hADAR2.

[0037] In the present application, the term “target RNA”, also referred to as “targeted RNA” or “Target RNA”, can be used interchangeably in the present application, and generally refers to an RNA containing a target site. In the present application, the target RNA can be pre-mRNA, mRNA, rRNA, tRNA, Inc-RNA, snRNA, snoRNA and microRNA, etc. Mutation of certain nucleotide sites can lead to different types of functional differences of the target RNA, such as abnormal splicing, alternative splicing of RNA, truncation, extension, misfolding of protein, etc.

[0038] In the present application, the term “target site”, also referred to as “editing site”, can be used interchangeably in the present application, and generally refers to a site in RNA editing directed by ASO or agRNA. In the present application, the target site can be adenosine.

[0039] In the present application, the term “targeting region”, also referred to as “targeting region”, “targeting domain”, can be used interchangeably in the present application, and generally refers to a partial region in ASO or agRNA, which is complementary to the region containing the target site in the target RNA.

[0040] In the present application, the term “recruiting region”, also referred to as “recruiting region”, “recruiting domain”, can be used interchangeably in the present application, and generally refers to a partial region in ASO or agRNA, which can bind and / or recruit RNA editing entity. In the present application, the recruiting region is not complementary to the region containing the target site in the target RNA. In the present application, the recruiting region has no stem-loop structure.

[0041] In the present application, the term “downstream” refers to further extending the sequence in the 3' direction; the term “upstream” refers to the opposite.

[0042] In the present application, the term “complementary pairing” can be used interchangeably with “complementary” and “pairing”, and generally refers to Watson-Crick or Hoogsteen base pairing between nucleotide units of nucleic acid molecules. In the present application, base pairing can refer to A-T, C-G, T*A / T, C*G / C. In the present application, complementary pairing can be complete complementary pairing, or there can be one or more bulges, wobbles, deletions, and / or mismatches between nucleic acid molecules. In the present application, ASO or agRNA can form a double-stranded complex with target RNA through complementary pairing.

[0043] In the present application, the term "bulge" generally refers to a region in which the upstream and downstream bases of the bulge region are complementary to the target RNA strand and the corresponding two bases of the target RNA strand are consecutive. In the present application, the term "wobble" generally refers to a G-U pairing. In the present application, the term "deletion" generally refers to a region in which the upstream and downstream bases of the deletion region are consecutive and there are corresponding number of bases in the target RNA strand at the deletion region. In the present application, the term "mismatch" generally refers to a pair of nucleotides in a double-stranded RNA complex that are not paired according to the Watson-Crick base pairing rules. The type of mismatch can be one of A-A, A-G, A-C, U-U, U-C, G-G, G-A, C-A, C-C, C-U, I-C, I-U, I-G, I-A.

[0044] In the present application, the term "perfect complementarity" generally refers to a nucleotide unit of a nucleic acid molecule that only exists strict Watson-Crick or Hoogsteen base pairing. Perfect complementarity does not exist bulge, wobble, deletion, and / or mismatch.

[0045] In the present application, the term "modification" generally refers to modification of a natural or synthetic component. The modification can include modification of a base, modification of a nucleoside, modification of a sugar, modification of a nucleotide linkage; can include chemical modification and non-chemical modification. In the present application, the modification can include LNA, UNA, 2'-MOE, 2'-OMe, 2'-F, phosphorothioate modification, base substitution modification (thymine, 5-methylcytosine and inosine), etc. LNA refers to a modification in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom. UNA refers to a modification in which the ribose ring lacks a C2' to C3' chemical bond. 2'-MOE refers to a modification in which the hydrogen on the 2'-hydroxyl of a ribonucleotide is replaced with a methoxyethyl group. 2'-OMe refers to a modification in which the hydrogen on the 2'-hydroxyl of a ribonucleotide is replaced with a methoxy group. 2'-F refers to a modification in which the hydrogen on the 2'-hydroxyl of a ribonucleotide is replaced with a fluorine. Phosphorothioate modification refers to a modification in which one of the non-bridging oxygen atoms in the original phosphate linkage is replaced with a sulfur atom. Base substitution modification refers to a modification in which thymine, 5-methylcytosine and inosine are used to replace uracil, cytosine and guanine nucleosides in the original sequence.

[0046] In the present application, the term "alkyl" refers to a branched or unbranched saturated or unsaturated hydrocarbon group of 1-14 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, and the like. The alkyl group can be cyclic or acyclic, such as cyclopropyl, cyclobutyl, cyclohexyl. In addition, the alkyl group can be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxyl, nitro, silyl, thio-oxo, or thiol. In addition, the alkyl group can be optionally further substituted. For example, the alkyl group can be substituted with halogen to form a haloalkyl or halogenated alkyl group, which can be substituted with one or more halogen, which refers to fluorine, chlorine, bromine, or iodine.

[0047] In the present application, the term "cell" generally includes prokaryotic cells and eukaryotic cells. A nucleic acid can be transfected into a cell, a plasmid can be propagated in a prokaryotic cell, and a nucleic acid can be expressed to encode a polypeptide in a eukaryotic cell. For example, a cell can include an agRNA and / or a delivery vector. A cell can be a cell from any organ, such as skin, lung, heart, kidney, liver, pancreas, intestine, muscle, gland, eye, brain, blood, and the like. For example, a cell can be a human cell or a mouse cell. For example, a cell can be an immune cell. For example, an immune cell can be a T cell, a B cell, a natural killer cell (NK cell), a macrophage, an NKT cell, a monocyte, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell, and / or a pluripotent stem cell. For example, an immune cell can be a T cell.

[0048] In the present application, the term "delivery vector" generally refers to a vector that delivers one or more nucleotides to a cell. The vector can include a viral vector, a non-viral vector. The viral vector can include a lentivirus (LV) vector, an adenovirus (AdV) vector, an adeno-associated virus (AAV) vector, and the like. The non-viral vector can include a liposome, a molecular conjugate, a polymer, a complex vector, a nanoparticle vector, and the like. In the present application, the delivery vector can deliver an agRNA or an isolated nucleic acid molecule described in the present application into a cell.

[0049] In the present application, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to an individual. For example, the pharmaceutical composition can include the agRNA, the delivery vector, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0050] In the present application, the term "pharmaceutically acceptable carrier" generally includes a pharmaceutically acceptable vehicle, excipient, or stabilizer, which is nontoxic to the cells or mammals being exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers can include suitable substances.

[0051] In the present application, the terms "adenine", "guanine", "cytosine", "thymine", "uracil", and "hypoxanthine" refer to the nucleobases themselves. The terms "adenosine", "guanosine", "cytidine", "thymidine", "uridine", and "inosine" refer to the nucleobases linked to a sugar moiety such as ribose or deoxyribose. The term "nucleoside" refers to a nucleobase linked to a sugar moiety such as ribose or deoxyribose.

[0052] In the present application, the term "nucleotide" refers to a respective nucleobase-ribosyl-phosphate or nucleobase-deoxyribosyl-phosphate or nucleobase-modified-sugar- phosphate. In the present application, the terms "adenosine" and "adenine" (abbreviated "A"), "guanosine" and "guanine" (abbreviated "G"), "cytidine" and "cytosine" (abbreviated "C"), "uridine" and "uracil" (abbreviated "U"), "thymidine" and "thymine" (abbreviated "T"), "inosine" and "hypoxanthine" (abbreviated "I"), are used interchangeably.

[0053] In the present application, the term "in vitro" refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in a cell culture, and the like, rather than within an organism (e.g., an animal, a plant, and / or a microorganism).

[0054] In the present application, the term "in vivo" refers to events that occur within an organism (e.g., an animal, a plant, and / or a microorganism).

[0055] In the present application, the term "and / or" should be understood to mean either of the items or both of the items.

[0056] In the present application, the term "comprising" generally means including, but not limited to, the specifically recited components. In certain situations, "comprising" also encompasses only including the specifically recited components. For example, comprising also means "consisting of" in some contexts.

[0057] In the present application, the term "about" generally means within 0.5-10% above or below the indicated numerical value, for example, within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the indicated numerical value.

[0058] In the present application, the term "comprising" generally means including, encompassing, containing or comprising, and in some cases, also means "consisting of".

[0059] DETAILED DESCRIPTION

[0060] Antisense Oligonucleotide guide RNA (agRNA)

[0061] In one aspect, the present application provides an Antisense Oligonucleotide guide RNA (agRNA) for RNA editing, the agRNA comprising: (1) a targeting region that is complementary to a target RNA; and (2) a recruiting region that is complementary to the target RNA and has no stem-loop structure, the recruiting region being capable of binding and / or recruiting an editing entity.

[0062] In the present application, a plurality of target sites of the target RNA can be edited by the agRNA, the number of the sites can be 2 or more. In the present application, the expression level of a gene can be regulated by the agRNA. For example, the expression level of one or more genes can be increased by the agRNA. In the present application, a plurality of editing sites of the target RNA can be edited by the agRNA and the expression level of a plurality of genes can be regulated. Synergy can exist between the two functions.

[0063] In the present application, the agRNA can comprise a plurality of targeting regions, the plurality of targeting regions can bind to a plurality of target sites on the target RNA respectively, and the plurality of targeting regions can be discontinuous. In the present application, the agRNA can comprise a plurality of recruiting regions, the plurality of recruiting regions do not bind to the target sites on the target RNA, and the plurality of recruiting regions can be discontinuous.

[0064] In the present application, the agRNA can comprise one targeting region and one recruiting region.

[0065] In the present application, the agRNA can have different forms. For example, it can be a linear agRNA molecule, it can be a circular agRNA molecule, it can have no chemical modification, or it can have one or more same or different chemical modifications.

[0066] In the present application, the agRNA can be synthesized by any means known in the art. For example, it can be synthesized by chemical synthesis, it can be expressed by a vector, or it can be expressed by a plasmid.

[0067] In the present application, the agRNA for RNA editing can be fully complementary to the target RNA, can be complementary to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges. The length and amount of the complementary pairing can vary depending on the target RNA, but can be routinely determined by one of ordinary skill in the art. Generally, longer sequences provide more specificity, and thus less off-target effects (e.g., by non-specific binding).

[0068] Targeting region and recruiting region

[0069] The agRNA provided in the present application comprises a targeting region, which can be complementary to a portion of the target RNA containing the target site. The agRNA can recognize and bind to the target RNA through the targeting region.

[0070] In the present application, the targeting region can be fully complementary to the target RNA, can be complementary to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges, can not have chemical modifications, and can have one or more same or different chemical modifications.

[0071] In the present application, the length of the targeting region can be greater than 6 nt. For example, the length of the targeting region can be 6-10 nt, 10-15 nt, 15-20 nt, 20-25 nt, 20-30 nt, 20-40 nt, 20-45 nt, 30-35 nt, 30-40 nt, 30-45 nt, 30-50 nt, 40-45 nt, 45-50 nt. For example, the length of the targeting region can be 5 nt, 10 nt, 15 nt, 20 nt, 25 nt.

[0072] The agRNA provided in the present application comprises a recruiting region, which can not be complementary to a portion of the target RNA containing the target site, and the recruiting region does not contain a stem-loop structure. The agRNA can bind and / or recruit editing entities through the recruiting region.

[0073] In the present application, the recruiting region can be fully complementary to the target RNA, can be complementary to the target RNA with one or more mismatches, wobbles, deletions, and / or bulges, can not have chemical modifications, and can have one or more same or different chemical modifications.

[0074] In the present application, the length of the recruiting region can be greater than 10 nt. For example, the length of the targeting region can be 10-15 nt, 15-20 nt, 20-25 nt, 20-30 nt, 20-40 nt, 20-45 nt, 30-35 nt, 30-40 nt, 30-45 nt, 30-50 nt, 40-45 nt, 45-50 nt. For example, the length of the targeting region can be 10 nt, 15 nt, 20 nt, 25 nt, 30 nt.

[0075] In the present application, the targeting region can be located upstream of the recruiting region, downstream of the recruiting region, directly connected to the recruiting region, or connected to the recruiting region through a linker.

[0076] In the present application, after the targeting region and the recruiting region bind to the target RNA, there can be no spacing, or there can be spacing, which can vary depending on the target RNA. For example, the spacing can be greater than 5 nt. For example, the spacing after binding can be 5 nt, 9 nt, 26 nt, 30 nt, 379 nt or more.

[0077] In the present application, the principle of complementary pairing is Watson-Crick or Hoogsteen base pairing. For example, the complementary pairing can refer to A-T, C-G, T*A / T (referring to protonated T can pair with A in A-T base pair), C*G / C (referring to protonated C can pair with G in G-C base pair). For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) base mismatches. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) wobbles. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) deletions. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, etc.) bulges.

[0078] For example, the complementarity between the targeting region, the recruiting region, and the target RNA is about 50-100% (e.g., about 50-80%, 50-85%, 50-90%, 50-95%, 60-80%, 60-85%, 60-90%, 60-95%, 60-100%, 65-80%, 65-85%, 65-90%, 65-95%, 65-100%, 70-80%, 70-85%, 70-90%, 70-95%, 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-100%, 80-85%, 80-90%, 80-95%, 80-100%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.). For example, the complementarity is at least about 60%. In some embodiments, the complementarity is at least about 65%. For example, the complementarity is at least about 70%. For example, the complementarity is at least about 75%. For example, the complementarity is at least about 80%. For example, the complementarity is at least about 85%. For example, the complementarity is at least about 90%. For example, the complementarity is at least about 95%.

[0079] In some embodiments, the targeting region alone can not produce the effect of editing the RNA. In some embodiments, the recruiting region alone can not produce the effect of editing the RNA.

[0080] Chemical modification

[0081] In some embodiments, the agRNA, the targeting region, the recruiting region can have one or more modifications.

[0082] For example, the modification can be a nucleobase modification. The modified nucleobase can have at least one function of a nucleobase. For example, capable of base pairing or forming Π-Π stacking interaction with upstream and downstream bases. For example, the modified nucleobase can be A, T, C, G, U, or 5-methyl-C (5mC). For example, the base can be modified by substitution, which can be DNA base substitution and / or nucleoside analog substitution (such as inosine). For example, the base can be modified by isomerization of glycosidic bond connection site (instead of natural N9 / N1 site), which includes N7 site of purine, N3 site of pyrimidine connection (such as isoU), or pyrimidine 5 / 6 carbon glycosidic bond connection (such as pseudouridine), etc. For example, the base can be modified by oxidation, which includes 8-oxo-purine, 6-oxo-pyrimidine, etc. For example, the base can be modified by alkylation or halogenation, which includes, for example, 5-halogen / alkyl pyrimidine, 8-halogen / alkyl purine, and single or combined modification of these modifications. For example, in the agRNA, the modified nucleobases can account for about 5%-100%, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.

[0083] For example, the modification can be a ribose modification. The modified sugar can have the spatial arrangement, electronic properties, or some other physico-chemical properties of unmodified ribose. For example, the modified sugar is a substituted ribose or deoxyribose. For example, the modified sugar is a substituted or unsubstituted arabinose (ANA), threose (TNA), hexitol (HNA), cyclopentane (CENA), cyclohexane (CHNA), or a modified sugar analog. Non-limiting examples of modified sugar analogs include derivatives, isomers, etc. of sugars, non-limiting examples of derivatives of sugars include 2’-substitution, 3’-substitution, 4’-substitution, 5’-substitution, and single or combination modifications of these modifications, etc. such as 2’-O-alkyl (e.g., 2’-O-Me), 2’F, 2’-O-methoxyethyl (2’-MOE), 2’-O-[2-(methylamino)-2-oxoethyl] (2’-O-NMA), 2’-formamide, 4’-O-methyl, 2’,4’-difluoro, 2’,2”-difluoro, etc.; also including open ring modifications (such as GNA, UNA), bicyclic modifications (such as LNA, cEt, BNA, bcDNA), tricyclic modifications (such as tcDNA), or INV modifications (such as INV at C1’ position alpha or beta), and derivatives or isomers thereof, etc. For example, the agRNA can comprise one or more modified sugars. For example, in the agRNA, the modified sugars can comprise about 5%-100%, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, etc.

[0084] For example, the modification can be a backbone modification, which includes a phosphorothioate backbone modification, an amide backbone modification, a cycloazole backbone modification (e.g., a 1,2,3-triazole ring formed by the reaction of an azide with an alkyne); the phosphorothioate backbone modification includes a phosphorothioate modification (i.e., replacing the non-bridging oxygen atom of a nucleotide phosphodiester linkage with a sulfur atom), a methyl phosphonamidate modification (i.e., replacing the non-bridging oxygen atom of a nucleotide phosphodiester linkage with a methyl phosphonamido group), a guanidinyl phosphoramidate modification (i.e., replacing the non-bridging oxygen atom of a nucleotide phosphodiester linkage with a guanidinyl or substituted guanidinyl group), a methylphosphonate modification (i.e., replacing the non-bridging oxygen atom of a nucleotide phosphodiester linkage with a methyl group).

[0085] Linker

[0086] In another aspect, the agRNA provided herein comprises a linker.

[0087] In the present application, the linker can be one or more of unmodified nucleotides, modified nucleotides, oligopeptides, and any other chemical linker. In the present application, the agRNA can comprise one or more linkers which can be the same or different.

[0088] For example, the linker can comprise one or more nucleotides, oligopeptides, or any other chemical linker, which can be conventional (phosphodiester) or modified (e.g., phosphorothioate). For example, the linker can be one or more of an alkyl unit, annealed double-stranded RNA, and RNA base sequence.

[0089] For example, the chemical linker can be one or more of PEG2, PEG6, C3, C6, C9.

[0090] For example, the RNA base sequence can be one or more of AAA, AAAC, AACAA, AAAACAAAA, and AAACAAACAAACAAA.

[0091] For example, the annealed double-stranded RNA can be 5-50 nt in length. For example, the annealed double-stranded RNA can be 5-15 nt, 5-25 nt, 5-35 nt, 5-25 nt, 5-35 nt, 5-40 nt, 5-35 nt, 5-40 nt, 5-45 nt in length. For example, the RNA base sequence as a linker can be designed and validated by different agRNA sequences.

[0092] Editing entity

[0093] In the present application, the editing entity is typically a protein in nature, such as the ADAR enzymes found in metazoans, including mammals. The editing entity can also comprise a complex of a nucleic acid and a protein or peptide, such as a ribonucleoprotein. The editing enzyme can comprise or consist of a nucleic acid only, such as a ribozyme. All of these editing entities are included in the present application, as long as they are recruited by the agRNA according to the present application.

[0094] For example, the editing entity can be an enzyme, can be an adenosine deaminase or a cytidine deaminase, can be an adenosine deaminase can be a human ADAR, hADAR1 and hADAR2, including any isoforms thereof, such as hADAR1 pi 10 and pi 50.

[0095] Target RNA

[0096] In the present application, the agRNA described in the present application can be used to make changes in a target RNA sequence, the agRNA is capable of targeting a site to be edited and recruiting an RNA editing entity present in the cell to cause an editing reaction. The editing reaction can be adenosine deamination and cytidine deamination, respectively converting adenosine into inosine and cytidine into uridine. These changes can be in the 5’ or 3’ untranslated region, (cryptic) splice site, exon, intron, and generally any region of the target RNA that affects the stability, structure or functionality of the RNA. The target RNA sequence can contain a mutation that can be desirable to correct or change. Alternatively, the target RNA is intentionally mutated to produce an altered phenotype that was not previously mutated.

[0097] In the present application, the target RNA can be any cellular or viral RNA sequence, can be a pre-mRNA, mRNA, rRNA, tRNA, Inc-RNA, snRNA, snoRNA and microRNA, but more typically a pre-mRNA or mRNA with protein coding functionality.

[0098] Method of RNA editing

[0099] In another aspect, the present application provides a method of RNA editing, which can comprise the use of the agRNA described. The method can be used to edit a target site of a target RNA, the method can be performed in vivo or in vitro. In the present application, the agRNA described can be introduced into a cell comprising a target RNA.

[0100] Cell

[0101] In another aspect, the present application provides a cell, which can comprise the agRNA, plasmid, and / or delivery vector described in the present application.

[0102] For example, the cell can be a mammalian cell, such as a human subject, a domestic animal, an experimental animal, such as a mouse or a rat cell. For example, the cell can be a human cell, the cell can be an established cell line.

[0103] For example, the cell is a cell having endogenous expression of ADAR. For example, the manipulation of the cell can be performed in vivo. For example, the cell can be isolated from the body, or a cell line can be cultured, and the manipulation can be performed in vitro.

[0104] Pharmaceutical composition, delivery of agRNA, application

[0105] In another aspect, the present application provides a pharmaceutical composition, which can comprise the agRNA, the delivery vector described in the present application, and optionally a pharmaceutically acceptable carrier.

[0106] For example, the pharmaceutical composition can be used for the treatment or prevention of a disease, such as gene therapy. For example, the pharmaceutical composition can also be used for diagnostic purposes, can not be used for diagnostic purposes, can be used for detecting parameters, can be used for laboratory purposes, such as in vitro experiments.

[0107] In the present application, the pharmaceutically acceptable carrier can include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, excipients, counterions, metal complexes, and / or nonionic surfactants, etc.

[0108] In another aspect, the present application provides a delivery vector, which can comprise the agRNA described in the present application, and the agRNA in the present application can be delivered to a cell.

[0109] In the present application, the delivery vector can comprise a lipid nanoparticle (LNP), a GalNAc conjugation modification, a polypeptide or antibody conjugation, and an exosome.

[0110] In the present application, the agRNA, the delivery vector, and the pharmaceutical composition described in the present application can be used by local administration or systemic administration.

[0111] In another aspect, the present application provides the agRNA, the delivery vector, the cell, the pharmaceutical composition, the use of the pharmaceutical in the preparation of a medicament for the prevention and / or treatment of a disease and / or a condition.

[0112] Without being bound by any theory, the following examples are merely intended to illustrate the fusion protein, the preparation method, and the use of the present application, and are not intended to limit the scope of the present application.

[0113] Examples

[0114] Materials and methods

[0115] Unless otherwise indicated, the reagents, methods, and apparatuses employed in the present application are of a type commonly employed in the art.

[0116] Unless otherwise indicated, the reagents and materials used in the following examples are commercially available.

[0117] 1. agRNA synthesis

[0118] In some embodiments, the agRNA is a chemically synthesized sequence with specific modifications, wherein the modifications include but are not limited to ribose modifications and backbone modifications, and the synthesis method is conventional solid-phase synthesis.

[0119] 2. Cell culture and transfection

[0120] The operating table is disinfected according to the conventional cell culture operation method, the cells are washed with PBS, then 0.25% trypsin digestion is added, the serum-containing medium is added to terminate, then the cells are counted and inoculated into the corresponding culture plate for subsequent transfection; the transfection method is performed according to the operation instruction of the transfection reagent.

[0121] 3. Editing efficiency determination

[0122] The total RNA of the cells can be extracted 48 hours after transfection, and then the RNA is subjected to reverse transcription and PCR amplification to obtain the upstream and downstream fragments of the editing site for first-generation sequencing. The editing efficiency is evaluated according to the sequencing file, and the EditR software (https: / / moriaritylab.shinyapps.io / editr_v10 / ) is used for calculation. The editing level at the editing site is calculated as 100*(1-A peak / A+T+G+C peak sum).

[0123] Example 1: Target region and recruitment region are connected by linker to improve editing efficiency

[0124] This example uses the strategy disclosed in the present application to design agRNA to edit ACTB, GAPDH and OGT genes. The design strategy of the present application is to add a linker to the target region sequence and the recruitment region sequence, where both the target region sequence and the recruitment region sequence bind to the target RNA. In this example, the length of the target region sequence is 15 nt, and the length of the recruitment region is 25 nt. The distance between the target region sequence and the recruitment region sequence after binding to the target RNA on the ACTB gene is 30 nt, and the distance on the GAPDH and OGT genes is 5 nt and 26 nt, respectively. The editing effect of the design strategy of the present application on the target site was tested in two cell lines, where HEK293 cells are ADAR1 overexpressing cell lines, and Hela cells are normal cell lines. Different sequences were transfected into the two cell lines according to the method described above, one was the target region sequence alone, one was the recruitment region sequence alone, one was the mixed target region sequence and recruitment region sequence co-transfected, and the last one was the design strategy of the present application using a linker to connect the two region sequences into one.

[0125] The results show that:

[0126] The editing site on the ACTB gene has no editing effect when the target region sequence or the recruitment region sequence is transfected alone, and after mixed transfection, there is more than 20% editing efficiency in Hela cells and 40% editing level in HEK293 cells, but after connecting the two domain sequences into one through the linker, the editing level in both cell lines is more than 20% higher. On the other two genes GAPDH and OGT, it is proved that the editing efficiency can be improved after connecting the linker, the difference is that the OGT gene has no editing effect when the two domain sequences are co-transfected in Hela cells, but it can be edited after the linker is connected. In addition, the recruitment region sequence was modified to introduce mismatches on the OGT gene, and the results showed that the recruitment region sequence also has recruitment effect when forming a non-completely complementary structure with the target RNA.

[0127] Example 2 Different lengths of target region and recruitment region improve editing efficiency through linker

[0128] Based on the data of Example 1, this example designs different length combinations of target region and recruitment region sequences, and tests them using ACTB gene as an example. The length of the target region sequence in this example is 10 nt, and the length of the recruitment region sequence is 20 nt and connected by a linker. The target region sequence, the recruitment region sequence, the mixed sequence of the target region and the recruitment region, and the single sequence connected by the linker were transfected into Hela cells and HEK293 cells, respectively.

[0129] The results show that:

[0130] In addition to the agRNA connected by the linker, other conditions cannot be edited under different length combinations, which further reflects the improvement effect of the design of the present application, wherein about 15% editing in Hela cells has more than 20% editing efficiency in HEK293 cells.

[0131] Example 3 Different types of linkers can improve editing efficiency

[0132] The experimental data described above show that the agRNA connected by the linker has an improvement effect on editing, so this embodiment tests other types of linkers on the basis of PEG2 linker and tests their effects on editing efficiency. The types of linkers tested include PEG6, C3, C6, and C9, and different linkers are added to sequence synthesis by solid-phase synthesis. In this embodiment, ACTB gene is taken as an example, and the length of the target region sequence and the recruitment region sequence is 15 nt and 25 nt, respectively.

[0133] The results show that:

[0134] In Hela and HEK293 cells, agRNA connected by different types of linkers has a significant improvement effect compared with the method of mixing two region sequences. The method of mixing two sequences has about 20% editing in Hela cells, while the editing efficiency after connecting different linkers is increased to about 65% to 70%. In HEK293 cells, the method of mixing two sequences can achieve about 25% editing, but after connecting the linker, it can achieve about 60% editing, which has a significant improvement effect.

[0135] Example 4 Fixed recruitment region sliding target region to different positions can achieve high editing efficiency

[0136] The design strategy in the prior art with a recruitment region is a sequence that can form a specific double-stranded structure, which is formed inside the guide RNA or between two sequences. The present application is formed by a sequence binding to the target RNA. In this embodiment, ACTB gene is taken as an example, and the position of the fixed 25 nt length recruitment region is tested for editing ability under different length intervals between the target region sequence. The editing effect of specific bases A (site2, site3 and site4) in the target region with 9 nt, 5 nt and 379 nt intervals is tested in two cell lines.

[0137] The results show that:

[0138] The agRNA designed according to the present application can achieve effective high editing levels at these sites, for example, at site 2, neither the sequence targeting the region nor the sequence recruiting the region has editing effect, and after being connected by the linker, about 50% editing is achieved in Hela cells, and more than 40% editing is achieved in HEK293 cells. Site 3 can achieve 45% to 50% editing effect in the two cell lines, and site 4 can achieve about 70% editing in the two cells.

[0139] Example 5 agRNA connected by a linker can edit a reporter gene with a specific secondary structure

[0140] The RNA in cells has various intramolecular and intermolecular folding conditions, so the secondary structure of the RNA is diverse, and the traditional guide RNA design will be difficult to edit the target site under some specific structure conditions. This embodiment designs a reporter gene to test whether the design of the present application can edit the target RNA with a specific structure. The design of the reporter gene is to add the upstream and downstream fragments of the editing site after the stop codon of the green fluorescent protein GFP gene, and the fragment is followed by a sequence that can form a 17bp double strand, and finally a fragment containing the recruiting region sequence. These fragments are constructed into a plasmid expression vector. The constructed vector is first transfected into two cell lines, and 6 hours later, the corresponding agRNA is transfected, and the control group is the mixed transfection of the sequence targeting the region and the sequence recruiting the region, and the experimental group is the agRNA designed by the present application.

[0141] The results show that:

[0142] The editing of the reporter gene by the design of the present application can reach 80% in Hela cells and 60% in HEK293 cells, which are higher than the 40% and 20% editing levels without the linker.

[0143] Example 6 agRNA connected by a linker has a higher editing level on a reporter gene than the traditional design strategy

[0144] This example is based on the reporter gene test, comparing the effect of the design of the application with the existing design strategy. One of the existing design strategies is a continuous single-stranded sequence that binds to the target RNA for editing. The other is a targeting sequence that binds to the target RNA and also forms a double-stranded recruitment structure. The recruitment sequence does not bind to the target RNA. The first traditional design is a continuous single-stranded sequence, which is the same as the targeting region sequence of the design of the application and extends to 40 nt in length. The second traditional design is the same as the design of the application, but its recruitment region selects the best published editing structure (source patent WO2022026928A1). First, the reporter gene vector is transferred into two cell lines for 6 hours, and then the two traditional design strategies and the design of the application are transfected respectively.

[0145] The results show that:

[0146] The design of the continuous single-stranded structure cannot be edited in Hela cells, and can achieve about 10% editing in HEK293 cells. The second traditional design strategy has about 65% editing in Hela cells and about 50% editing in HEK293 cells. The design of the application can achieve 74% editing in Hela cells and 69% editing in HEK293 cells, indicating that the editing effect of the reporter gene of the design of the application is higher than that of the two traditional design strategies.

[0147] Sequences used in this application

Claims

1. An ADAR guide RNA (agRNA) for RNA editing, the agRNA comprising: (1) a targeting region that is complementary paired to a target RNA; and (2) a recruiting region that is complementary paired to the target RNA and lacks a stem loop structure, the recruiting region being capable of binding and / or recruiting an editing entity.

2. The agRNA of claim 1, wherein the targeting region is complementary paired to a portion of the target RNA that contains a target site.

3. The agRNA of any one of claims 1-2, wherein the targeting region is greater than 6 nt in length.

4. The agRNA of any one of claims 1-3, wherein the targeting region is 10-15 nt in length.

5. The agRNA of any one of claims 1-4, wherein the recruiting region is not complementary paired to a portion of the target RNA that contains a target site.

6. The agRNA of any one of claims 1-5, wherein the recruiting region is greater than 10 nt in length.

7. The agRNA of any one of claims 1-6, wherein the recruiting region is 20-25 nt in length.

8. The agRNA of any one of claims 1-7, wherein the editing entity comprises a deaminase activity.

9. The agRNA of any one of claims 1-8, wherein the editing entity comprises an adenosine deaminase acting on RNA (ADAR).

10. The agRNA of any one of claims 1-9, wherein the editing entity comprises hADAR1 and hADAR2.

11. The agRNA of any one of claims 1-10, wherein the target site is adenosine.

12. The agRNA of any one of claims 1-11, wherein the targeting region and the recruiting region are linked by a linker.

13. The agRNA of any one of claims 1-12, wherein the linker is optionally selected from the group consisting of: one or more unmodified nucleotides, one or more modified nucleotides, an oligopeptide, and any other chemical linker.

14. The agRNA of any one of claims 1-13, wherein the linker is optionally selected from the group consisting of: an alkyl, a PEG chain, an annealed double stranded RNA, and a sequence of RNA bases.

15. The agRNA of any one of claims 1-14, wherein the linker is: PEG2, PEG6, C3, C6, or C9.

16. The agRNA of any one of claims 1-15, wherein the agRNA comprises a chemical modification.

17. The agRNA of claim 16, wherein the chemical modification comprises one or more of a base modification, a sugar modification, a backbone modification, and a combination of these modifications.

18. The agRNA according to any one of claims 16-17, wherein the chemical modification is optionally selected from the group consisting of: LNA, UNA, 2’-MOE, 2’-F, 2’-OMe, base substitution modification (thymine, 5-methylcytosine and inosine), and phosphorothioated modification.

19. The agRNA according to any one of claims 1-18, wherein the agRNA is fully complementary paired to the target RNA.

20. The agRNA according to any one of claims 1-18, wherein the agRNA is complementary paired to the target RNA with one or more mismatches, wobbles, deletions and / or bulges.

21. The agRNA according to any one of claims 1-20, wherein the target RNA is optionally selected from the group consisting of: pre-mRNA, mRNA, rRNA, tRNA, Inc-RNA, snRNA, snoRNA and microRNA.

22. A delivery vehicle comprising the agRNA according to any one of claims 1-21.

23. A pharmaceutical composition comprising the agRNA according to any one of claims 1-21, and / or the delivery vehicle according to claim 21, and optionally a pharmaceutically acceptable carrier.

24. A plasmid for expressing the full length and / or partial sequence of the agRNA according to any one of claims 1-21.

25. A method for site-directed editing of a nucleotide in a target RNA in a eukaryotic cell, comprising using the agRNA according to any one of claims 1-21.

26. Use of the agRNA according to any one of claims 1-21, the delivery vehicle according to claim 22, and / or the pharmaceutical composition according to claim 23, in the manufacture of a medicament for the prevention and / or treatment of a disease and / or disorder.

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