Miniaturized hairpin RNAi triggers (mxRNAs) and methods of using them

Abstract

The present invention relates to novel RNAi triggers that can be chemically synthesized to regulate gene expression in animal cells and used to study various gene functions in the laboratory or as active ingredients in agricultural, veterinary, cosmetic and / or therapeutic applications. [Selected Figure] Figure 1

Description

Technical Field

[0001] The present invention relates to a novel RNAi trigger that is chemically synthesized, regulates gene expression in animal cells, and can be used to study various gene functions in the laboratory or as an active ingredient for agricultural, veterinary, cosmetic and / or therapeutic applications.

Background Art

[0002] RNA interference or RNAi is a biological phenomenon characterized by the ability of double-stranded RNA molecules to specifically downregulate individual genes in animals. Discovered in 1998 by Craig Mello and Andrew Fire, it was awarded the Nobel Prize in Physiology or Medicine in 2006 because it was expected to provide a new type of therapeutic agent. Since 2001, when it was shown that chemically synthesized RNAi triggers (small interfering RNAs or siRNAs) function in mammalian cell culture, siRNAs have been widely used in laboratories around the world to study various gene functions. The first RNAi drug, Onpattro™, was approved by the FDA in August 2018 to help patients with hereditary ATTR amyloidosis. Currently, many other RNAi drugs are being developed and tested in preclinical and clinical trials.

[0003] RNAi is promised to become one of the major new drug modalities, but there are specific issues associated with it. One of them is the high production cost of the active ingredient. Conventional RNAi triggers - small interfering RNAs (or siRNAs) - are composed of two 19-25 nt long oligonucleotides annealed to each other (a total of about 40-50 nucleotides). The production of such molecules requires sophisticated multi-step synthesis and, in some cases, extensive purification procedures, resulting in relatively high production costs. The first RNAi drug, Onpattro™, is offered at $450,000 per year per treatment, and one of the factors for such a high price is likely to be the production cost of the drug.

Summary of the Invention

[0004] The terms used in this specification are for the purpose of describing particular embodiments only and are not intended to limit the present invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated items. As used in this specification, the singular forms "a", "an", and "the" (original text) are intended to include the plural as well as the singular, unless the context clearly dictates otherwise. It will be further understood that the terms "comprise" and / or "comprising" as used in this specification identify the presence of the described features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0005] Unless otherwise defined, all terms (including technical and scientific terms) used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art and this disclosure, and should not be interpreted in an idealized or overly formal sense unless explicitly defined in this specification.

[0006] In describing the present invention, it will be understood that several features, steps, operations, elements, and / or components are disclosed. Each of these has its own individual advantages and each may also be used in combination with one or more, or in some cases all, of the other disclosed features, steps, operations, elements, and / or components. Therefore, for the sake of clarity, this specification avoids repeating in an unnecessary way all possible combinations of the individual features, steps, operations, elements, and / or components. Nevertheless, it should be read with the understanding that such combinations are fully within the scope of the present invention.

[0007] A new miniaturized hairpin RNAi trigger (mxRNA) and methods of using the same are discussed herein. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the invention. It will be apparent to one skilled in the art, however, that the invention may be practiced without these specific details.

[0008] This disclosure should be considered as illustrative of the invention and is not intended to limit the invention to the specific embodiments illustrated in the figures or described below.

[0009] The invention will now be described by reference to the accompanying drawings that illustrate specific embodiments. A miniaturized hairpin RNAi trigger molecule (mxRNA) is introduced that includes the following components: 1) a 5' segment of the hairpin stem (segment A), 2) a hairpin loop (segment B), 3) a 3' segment of the hairpin stem (segment C), Here, 1) the total length of (A) + (B) + (C) is 17 to 40 nucleotides, 2) (B) is 0 to 10 nucleotides, 3) (A) is 0 to 4 nucleotides longer or 0 to 4 nucleotides shorter than (B) 4) 17 or more nucleotides from the 5' end of the molecule are complementary to a target RNA, e.g., mRNA, IncRNA, and / or other RNA molecules, Also (optionally), internal nucleotides of either or both of the single-stranded region or double-stranded region are chemically modified in the sugar and / or base and / or phosphodiester moiety of the molecule, e.g., by 2'OMe, 2'F, LNA, PMO, phosphorothioate (PS, PS2), or other chemical modifications, to improve the desired properties of the molecule (e.g., to increase stability against intracellular and / or extracellular nucleases), Cap the ends of the molecule, for example with vinylphosphonate, inverted nucleotides, or other modifications, or chemically modify to improve the desired properties of the molecule (e.g., to increase stability against intracellular and / or extracellular nucleases), Conjugate the mxRNA molecule to various delivery moieties such as cholesterol, carbohydrates (GalNAc, others), aptamers, peptides, small molecules, and / or others to direct and facilitate extracellular and intracellular delivery of the molecule.

[0010] According to the present invention, there is also provided a conjugate for regulating, preferably inhibiting, the expression of a target gene in a cell, the conjugate comprising a nucleic acid bound to one or more ligands, the nucleic acid preferably not being a substrate for Dicer,

[0011] comprising first, second, and third nucleic acid portions,

[0012] The first portion (i) is at least partially complementary to at least a portion of the RNA transcribed from the target gene and (ii) has a 5' to 3' directionality, thereby defining 5' and 3' regions of the first portion,

[0013] The second portion (i) is at least partially complementary to the first portion and (ii) has a 5' to 3' directionality, thereby defining 5' and 3' regions of the second portion,

[0014] The first and second portions dimerize to form at least a partially complementary double strand,

[0015] The third nucleic acid portion links the 3' region of the first portion to the 5' region of the second portion.

[0016] The conjugate according to the present invention comprises a third nucleic acid moiety that is at least partially complementary to at least a portion of the RNA transcribed from the target gene. Further, the conjugate according to the present invention can comprise a second nucleic acid moiety that is at least partially complementary to at least a portion of the RNA transcribed from the target gene.

[0017] Preferably, the conjugate according to the present invention comprises one or more ligands conjugated to the second nucleic acid moiety. Suitably, the one or more ligands are conjugated to the 3' region of the second nucleic acid moiety. Alternatively, the one or more ligands are conjugated to the 3' region of the first nucleic acid moiety and / or the 5' region of the second nucleic acid moiety. Yet a further alternative is that the one or more ligands are conjugated to one or more regions intermediate the 5' and 3' regions of the first nucleic acid moiety and / or one or more regions intermediate the 5' and 3' regions of the second nucleic acid moiety. As yet a further alternative, the one or more ligands are conjugated to one or more regions of the third nucleic acid moiety.

[0018] Typically, the one or more ligands are any cell directing moiety such as a lipid, carbohydrate, aptamer, vitamin and / or peptide that binds to a specific target on the cell membrane or cell surface. In a preferred embodiment, the one or more ligands comprise one or more carbohydrates, for example, monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides. Even more preferably, the one or more carbohydrates comprise one or more galactose moieties, one or more lactose moieties, one or more N-acetyl-galactosamine moieties, and / or one or more mannose moieties, for example, one or more N-acetyl-galactosamine moieties, preferably two or three N-acetyl-galactosamine moieties.

[0019] One or more ligands can bind to a nucleic acid in a linear configuration or a branched configuration such that, for example, in a bifurcated or trifurcated configuration, or in a configuration based on a single ligand at different positions, the ligand binds to the nucleic acid.

[0020] The conjugate according to the present invention comprises a nucleic acid that is a single strand that dimerizes to thereby form a double strand in which the first and second portions are at least partially complementary. Typically, the nucleic acid is 17 to 40 nucleotides in length, preferably at least 20 nucleotides in length, or more preferably at least 25 nucleotides in length.

[0021] In the conjugate according to the present invention, the first nucleic acid portion is 7 to 20 nucleotides in length, preferably 10 to 18 nucleotides in length, more preferably less than 18 nucleotides in length. Similarly, in the conjugate according to the present invention, the second nucleic acid portion is 7 to 20 nucleotides in length, preferably 10 to 18 nucleotides in length, more preferably less than 18 nucleotides in length. Further, in the conjugate according to the present invention, the third nucleic acid portion is preferably 1 to 10 nucleotides in length, for example, 4 to 9 nucleotides in length, for example, 4, 5, 7, or 9 nucleotides in length.

[0022] The conjugate according to the present invention further comprises phosphorothioate or phosphorodithioate internucleotide linkages, for example, 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages. Typically, phosphorothioate or phosphorodithioate internucleotide linkages at one or more of the 5' and / or 3' regions of the first and / or second nucleic acid portions. In a preferred embodiment, the conjugate according to the present invention

[0023] Depending on the number of nucleotides present in the third nucleic acid moiety, at least 2, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably 10 phosphorothioate or phosphorodithioate internucleotide linkages between adjacent nucleotides of the third nucleic acid moiety are included. Further, the conjugate according to the invention can include phosphorothioate or phosphorodithioate internucleotide linkages between each adjacent nucleotide present in the third nucleic acid moiety. Further, the conjugate according to the invention can include phosphorothioate or phosphorodithioate internucleotide linkages that link the first nucleic acid moiety to the third nucleic acid moiety and / or the second nucleic acid moiety to the third nucleic acid moiety.

[0024] The conjugate according to the invention further includes at least one nucleotide of the modified first and / or second and / or third nucleic acid moieties. For example, in the conjugate according to the invention, one or more of the odd-numbered nucleotides starting from the 5' region of the first nucleic acid moiety are modified, and / or one or more of the even-numbered nucleotides starting from the 5' region of the first nucleic acid moiety are modified. Typically, the modification of the even-numbered nucleotides is a second modification different from the modification of the odd-numbered nucleotides. Typically, one or more of the odd-numbered nucleotides starting from the 3' region of the second nucleic acid moiety are modified by a modification different from the modification of the odd-numbered nucleotides of the first nucleic acid moiety.

[0025] Further features of the modification pattern can be as follows, but to avoid doubt, the following description does not limit the scope of the invention described herein: One or more of the even-numbered nucleotides starting from the 3' region of the second nucleic acid moiety are modified by a modification different from the modification of the odd-numbered nucleotides of the second nucleic acid moiety, and / or At least one or more of the modified even nucleotides of the first nucleic acid moiety are adjacent to at least one or more of the differently modified odd nucleotides of the first nucleic acid moiety, and / or At least one or more of the modified even nucleotides of the second nucleic acid moiety are adjacent to at least one or more of the differently modified odd nucleotides of the second nucleic acid moiety, and / or A plurality of adjacent nucleotides of the first nucleic acid moiety are modified by a common modification, and / or A plurality of adjacent nucleotides of the second nucleic acid moiety are modified by a common modification, and / or When the third nucleic acid moiety links the 3' region of the first moiety to the 5' region of the second moiety, the plurality of adjacent generally modified nucleotides are typically 2 to 4 adjacent nucleotides, preferably 3 or 4 adjacent nucleotides, such that the plurality of adjacent generally modified nucleotides are located in the 5' region of the second nucleic acid moiety, and / or A plurality of adjacent generally modified nucleotides are located in the third nucleic acid region, and / or A plurality of odd nucleotides of the first and / or second nucleic acid moieties are typically modified such that the plurality of odd nucleotides are modified by a common modification, and / or A plurality of even nucleotides of the first and / or second nucleic acid moieties are typically modified by a second modification such that the plurality of even nucleotides are modified by a common second modification, and / or One or more modified nucleotides of the first nucleic acid moiety do not have a common modification present in the corresponding nucleotides of the double-stranded second nucleic acid moiety, and / or One or more modified nucleotides of the first nucleic acid moiety are shifted by at least one nucleotide relative to the generally modified nucleotides of the second nucleic acid moiety.

[0026] Typically, in the conjugates according to the present invention, the modification and / or the plurality of modifications are each individually a sugar, backbone or base modification, preferably selected from the group consisting of 3'-terminal deoxythymine, 2'-O-methyl, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification, morpholino modification, phosphoramidate modification, phosphorothioate or phosphorodithioate group modification, 5'-phosphate or 5'-phosphate mimic modification, and cholesteryl derivative or bisdecylamide group modification of dodecanoic acid.

[0027] The modification can be any one of locked nucleotides, abasic nucleotides, or unnatural bases containing nucleotides.

[0028] In a preferred embodiment, at least one modification is 2'-O-methyl. In a further preferred embodiment, at least one modification is 2'-F.

[0029] In a further preferred embodiment, any nucleotide downstream of the 2nd and 14th positions from the 1st nucleotide in the 5' region of the 1st nucleic acid moiety does not contain a 2'-O-methyl modification in the ribose moiety, and / or at any of the 9th to 11th positions downstream from the 1st nucleotide in the 5' region of the 1st nucleic acid moiety, the nucleotide of the 2nd nucleic acid moiety corresponding to the position of any of the nucleotides of the 1st nucleic acid moiety does not contain a 2'-O-methyl modification in the ribose moiety.

[0030] The conjugate according to the present invention preferably further comprises one or more unmodified nucleotides that can typically replace any of the aforementioned modified nucleotides. Such one or more unmodified nucleotides can be located in the 5' region of the 2nd nucleic acid moiety and / or can be located in the 3rd nucleic acid moiety at a proximal position to the 2nd nucleic acid moiety.

[0031] Preferably, one or more, preferably one, unmodified nucleotide is a nucleotide (or nucleotides) in the 5' region of the second nucleic acid moiety, typically a nucleotide of the second nucleic acid moiety directly attached to the third nucleic acid moiety, and / or a nucleotide (or nucleotides) of the third nucleic acid moiety proximate to the 5' region of the second nucleic acid moiety, typically a nucleotide of the third nucleic acid moiety directly attached to the second nucleic acid moiety, preferably representing any one of the 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, and / or 25th positions downstream from the first nucleotide in the 5' region of the first nucleic acid moiety, preferably any one of the 18th, 19th, 20th, and / or 21st positions downstream.

[0032] In the conjugate according to the present invention, typically all nucleotides other than the unmodified nucleotide, and / or a nucleotide at either the 2nd or 14th position downstream from the first nucleotide in the 5' region of the first nucleic acid moiety, and / or a nucleotide of the second nucleotide moiety corresponding in position to any one of the nucleotides of the first nucleic acid moiety at any one of the 9th to 11th positions downstream from the first nucleotide in the 5' region of the first nucleic acid moiety, contain a 2'-O-methyl modification on the ribose moiety.

[0033] In a preferred embodiment, all odd nucleotides of the first nucleic acid region starting from the 5' region of the first nucleic acid moiety are 2'-O-methyl modified, and all even nucleotides of the first nucleic acid region starting from the 5' region of the first nucleic acid moiety are 2'-F modified.

[0034] In certain embodiments, all odd nucleotides of the second nucleic acid region starting from the 3' region of the second nucleic acid portion, other than the unmodified nucleotide(s) of the second nucleic acid portion, are 2'-F modified, and all even nucleotides of the second nucleic acid region starting from the 3' region of the second nucleic acid portion are 2'-O-methyl modified. For example, a plurality of adjacent generally modified nucleotides of 2 to 4 adjacent nucleotides, preferably 3 or 4 adjacent nucleotides, are located downstream of the unmodified nucleotide(s) of the second nucleic acid portion, and for the remaining nucleotides of the second nucleic acid portion, all odd nucleotides of the second nucleic acid region starting from the 3' region of the second nucleic acid portion are

[0035] 2'-F modified, and all even nucleotides of the second nucleic acid region starting from the 3' region of the second nucleic acid portion are 2'-O-methyl modified.

[0036] In a further embodiment of the invention, in the conjugate described herein, the nucleotides of the third nucleic acid portion are modified in an alternating 2'-O-methyl, 2'-F pattern starting from a 2'-O-methyl modification adjacent to the 3' region of the first nucleic acid portion.

[0037] The conjugate according to the invention can further comprise at least one vinylphosphonate modification, for example at least one vinylphosphonate modification in the 5' region of the first nucleic acid portion.

[0038] In the conjugate according to the invention, at least one of the one or more nucleotides of the first nucleic acid portion and the second nucleic acid portion is an inverted nucleotide, which is linked to an adjacent nucleotide via the 3'-carbon of the nucleotide and the 3'-carbon of the adjacent nucleotide, and / or at least one of the one or more nucleotides of the first nucleic acid portion and the second nucleic acid portion is an inverted nucleotide, which is linked to an adjacent nucleotide via the 5'-carbon of the nucleotide and the 5'-carbon of the adjacent nucleotide.

[0039] The conjugate according to the present invention is one or more nucleotides in at least one of the 3'-regions of the first nucleic acid moiety and the second nucleic acid moiety, which are inverted nucleotides and are linked to an adjacent nucleotide via the 3'-carbon of the terminal nucleotide and the 3'-carbon of the adjacent nucleotide, and / or one or more nucleotides in at least one of the 5'-regions of the first nucleic acid moiety and the second nucleic acid moiety, which are inverted nucleotides and are linked to an adjacent nucleotide via the 5'-carbon of the terminal nucleotide and the 5'-carbon of the adjacent nucleotide, and / or one or more nucleotides in the middle of at least one of the 3'- and 5'-regions of the first nucleic acid moiety and the second nucleic acid moiety, wherein one or more nucleotides are inverted nucleotides and are linked to an adjacent nucleotide via the 3'-carbon of the terminal nucleotide and the 3'-carbon of the adjacent nucleotide, and / or one or more nucleotides in the middle of at least one of the 3'- and 5'-regions of the first nucleic acid moiety and the second nucleic acid moiety, which are inverted nucleotides and are linked to an adjacent nucleotide via the 5'-carbon of the terminal nucleotide and the 5'-carbon of the adjacent nucleotide, and / or one or more nucleotides of at least one of the third nucleic acid moieties, which are inverted nucleotides and are linked to an adjacent nucleotide via the 3'-carbon of the terminal nucleotide and the said 3'-carbon of the adjacent nucleotide, and / or one or more nucleotides of at least one of the third nucleic acid moieties, which are inverted nucleotides and are linked to an adjacent nucleotide via the 5'-carbon of the terminal nucleotide and the 5'-carbon of the adjacent nucleotide, and can further comprise. Typically, the 3'- and / or 5'-inverted nucleotides of the first and / or second strand are linked to an adjacent nucleotide via a phosphate group by a phosphodiester bond, or the 3'- and / or 5'-inverted nucleotides of the first and / or second strand are linked to an adjacent nucleotide via a phosphorothioate group, or the 3'- and / or 5'-inverted nucleotides of the first and / or second strand are linked to an adjacent nucleotide via a phosphorodithioate group.

[0040] The conjugate according to the present invention can have a blunt end at one end. Alternatively, the conjugate according to the present invention can include a first or second nucleic acid moiety having an overhang.

[0041] According to the present invention, there is further provided a homodimer RNA molecule comprising the two aforementioned nucleic acid molecules, wherein the nucleic acid molecules are bound together via complementary interactions, a first portion of the first molecule interacts with a second portion of the second molecule, and a third portion is present within each molecule that generates bulge structure intermediates of the first and second portions of the respective nucleic acid molecules.

[0042] The conjugate or homodimer RNA molecule and / or conjugate described herein are directed to a target RNA selected from at least one of mRNA, IncRNA, and / or other RNA molecules.

[0043] The present invention further includes: A composition comprising the conjugate or molecule described herein and a physiologically acceptable excipient, The conjugate or molecule described herein for use in the treatment of a disease or disorder, Use of the conjugate or molecule described herein in the manufacture of a medicament for treating a disease or disorder, A method for treating a disease or disorder, comprising administering the conjugate or molecule described herein to an individual in need thereof, for example, by subcutaneous or intravenous administration to the individual, Use of the conjugate or molecule described herein for use in research as a gene function analysis tool, A process for making the conjugate described herein.

[0044] Also provided by the present invention is a conjugate as described above, wherein the conjugate comprises a sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, and 18, and the linker and the trivalent GalNAc moiety are at the 3'-end of the nucleic acid moiety. For each of the sequences of SEQ ID NOs: 14, 15, 16, 17, and 18, these comprise first, second, and third nucleic acid moieties as follows, The first moiety begins at the start of each SEQ ID NO and (i) is at least partially complementary to at least a portion of the RNA (in this case, MAP4K4) transcribed from the target gene, and (ii) has a 5' to 3' directionality, thereby defining a 5'-region and a 3'-region of the first moiety, The second moiety (i) is at least partially complementary to the first moiety, and (ii) has a 5' to 3' directionality, thereby defining 5' and 3' regions of the second moiety, A third nucleic acid moiety links the 3'-region of the first moiety to the 5'-region of the second moiety, and in the sequence, For SEQ ID NO: 14: the first moiety comprises 14 nucleotides, the second moiety comprises 14 nucleotides, and the third moiety comprises 5 nucleotides, For SEQ ID NO: 15: the first moiety comprises 14 nucleotides, the second moiety comprises 14 nucleotides, and the third moiety comprises 4 nucleotides, For SEQ ID NO: 16: the first moiety comprises 12 nucleotides, the second moiety comprises 12 nucleotides, and the third moiety comprises 7 nucleotides, For SEQ ID NO: 17: the first moiety comprises 13 nucleotides, the second moiety comprises 13 nucleotides, and the third moiety comprises 4 nucleotides, For SEQ ID NO: 18: the first moiety comprises 10 nucleotides, the second moiety comprises 10 nucleotides, and the third moiety comprises 9 nucleotides.

[0045] For the sequences described above, the first and second moieties dimerize to form the at least partially complementary duplex described above.

[0046] Typically, such an array includes phosphorothioate or phosphorodithioate internucleotide linkages between each nucleotide of its third nucleic acid portion and / or unmodified nucleotides at positions 17, 18, 19, 20, 21, 22, 23, 24, and / or 25 from the 5' region of the first portion.

Brief Description of the Drawings

[0047]

Figure 1

[0048] Example 1 of FIG. 1 shows a schematic diagram of one of the smallest possible mxRNAs, where segment A is 7 nucleotides, segment B is 4 nucleotides, segment C is 7 nucleotides, and all 18 nucleotides are complementary to the target RNA.

[0049] Example 2 of FIG. 1 shows a schematic diagram of an mxRNA, where segment A is 14 nucleotides, segment B is 4 nucleotides, segment C is 14 nucleotides (total 32 nucleotides), 18 nt from the 5' end of the molecule is complementary to the target RNA (thick line), the triangle at the 5' end of the molecule represents a cap, such as a chemical moiety like vinylphosphonate, to increase resistance to nucleases, and a trivalent chemical moiety, such as GalNAc (line and circle), is conjugated to the 3' end of the molecule via a linker (wavy line) to facilitate delivery of the molecule to cells, such as hepatocytes in vitro and / or in vivo.

[0050] Example 3 of FIG. 1 shows a schematic diagram of mxRNA, where segment A is 18 nucleotides, segment B is 4 nucleotides, segment C is 18 nucleotides (total 40 nucleotides), the first 18 nucleotides from the 5' end of the molecule are complementary to the target RNA (thick line), the whole molecule is chemically modified with sugar modifications, such as 2'OMe and / or 2'F, to increase nuclease stability (not shown), the 2 nucleotides at each end of the molecule and the nucleotides within the loop are modified at the phosphodiester positions, such as phosphorothioate (star), to increase nuclease stability, and a delivery conjugate moiety (e.g., GalNAc, cholesterol, etc.) is attached to the single-stranded loop region of the molecule.

[0051] FIG. 1 illustrates three examples of mxRNA molecules as described above. The stem-and-loop configuration of Example 1 exemplifies one of the smallest versions of mxRNA - 18 nucleotides in length. Basically, the entire sequence of the molecule is complementary to the target RNA. In such an almost extreme case, it is understood that the target sequence needs to have relatively long (7 nucleotides) palindromic sequences separated by 4 nucleotides. In contrast, Example 3 shows a molecule having one of the largest (40 nucleotides in total) mxRNA stem-and-loop configurations. Such a configuration is similar to the structure of conventional shRNA, but there is an important difference in that it is highly unlikely to be processed intracellularly by the Dicer enzyme to generate conventional siRNA molecules (due to the single-stranded regions at the locations where Dicer is expected to cleave and / or due to chemical modifications that are likely to be used to stabilize the molecule against endonucleases). Example 2 shows an intermediate version (between the configurations shown in Example 1 and Example 3) of the mxRNA stem-and-loop configuration. Such a configuration does not impose constraints on the target sequence, but is much more compact than conventional siRNA and shRNA. It is understood that numerous other permutations in the stem-and-loop configuration design are possible.

[0052] Figure 1 also shows where specific chemical modifications and conjugations can be applied, particularly in Examples 2 and 3. In particular, any nucleotide (of the sugar, base, and / or phosphodiester bond) in the internal backbone of the molecule can be modified with various chemical modifications to improve the properties of the molecule (e.g., to increase stability against intracellular and extracellular nucleases). Furthermore, the ends of the molecule can be further enhanced by cap structures and chemical modifications. Various nucleic acid and non-nucleic acid moieties can also be conjugated to various parts of the mxRNA to add additional properties (e.g., enhanced and / or targeted delivery capabilities).

Figure 2

Figure 3

Figure 4

[0053] The mxRNA molecule can be chemically synthesized using conventional and / or advanced approaches and can be used as a research tool for studying various gene functions in the laboratory and / or as an active ingredient for agricultural, veterinary, cosmetic, and / or therapeutic applications. Aspects of the invention are demonstrated by the following non-limiting examples.

Examples

[0054] Example 1: Single-dose transfection in AML-12 cells An activity test of mxRNA against a conventional double-stranded siRNA construct directed against MAP4K4 was carried out. Hep3B cells were incubated in a 96-well plate at a density of 15,000 cells per well. The compounds tested in this study had a final concentration of 50 nM. Reverse transfection was performed using RNAiMax at 0.3 pL per well. In addition to the test compounds, two controls ((TTRPC) TTR-directed siRNA and (INTPC) aha-1-directed siRNA) were also used (Tables 3 and 4). The incubation time was 24 hours. Subsequently, mRNA was isolated and quantified using a bDNA assay (Quantigene 1.0 / 2.0). The readings were normalized to the GAPDH transcript, and the average value of quadruplicate replicates was determined. The value from mock-treated cells was set to 1.

[0055] An overview of the results obtained from this experiment is presented in Table 1 and Figure 2.

Table 1

[0056] Example 2: Single-dose direct incubation of GalNAc-conjugated compounds in primary cultured hepatocytes Primary cultured mouse hepatocytes (lot number MC830; Thermo Fisher Scientific) were incubated in a 96-well plate at a density of 60,000 cells per well. The compounds tested in this study were added at a final concentration of 500 nM. In addition to the test compounds, two controls ((XD-12171) TTR-directed siRNA and (XD-00033) aha-1-directed siRNA as negative controls) were also used (Tables 3 and 4). The direct incubation transfection (without transfection lipid) method was used. The duration of incubation was 72 hours. Subsequently, mRNA was isolated and quantified using a bDNA assay (Quantigene 1.0 / 2.0). The readings were normalized to GAPDH, and the average value of quadruplicate replicates was determined. The value from mock-treated cells was set to 1.

[0057] The summary of the results obtained from this experiment is presented in Table 2 and Figure 3. [Table 2]

[0058] Summary of the results from Examples 1 and 2 The results confirm that, as previously demonstrated by Lapierre et al, 2011, single-stranded oligonucleotide miniaturized hairpin structures (mxRNA) can cause target gene knockdown when used with transfection reagents and not blocked at the conjugate moiety.

[0059] It was demonstrated that mxRNA molecules conjugated with a bulky chemical moiety (GalNAc in this case) can still cause target gene knockdown when used with transfection reagents. Adding a conjugate to the 3'-end of the active strand may affect the ability of mxRNA to be recognized by the RNAi machinery, enter the RNA-induced silencing complex (RISC), and / or maintain activity within RISC, so this is a new and non-obvious finding.

[0060] Next, it was demonstrated that mxRNA conjugates (conjugates with GalNAc in this case) enter cells via receptor-mediated uptake and provide higher activity than conventional siRNAs targeting the exact same portion of the mRNA (e.g., mxRNA constructs C24, C25, C26 compared to conventional constructs C16, C17). This is a new discovery and without wishing to be bound by any particular theory, such improvement may be due to the smaller size of the mxRNA conjugate molecules compared to conventional siRNAs (approximately 42 nucleotides in total) (approximately 32 nucleotides in total).

[0061] Finally, the results showed that the use of diverse chemical modification patterns, including phosphodiester bond modifications (e.g., phosphorothioate modifications) and / or sugar modifications (e.g., at the 2’OH position), can further improve the performance of mxRNA.

Table 3-1

Table 3-2

Table 4-1

Table 4-2

Claims

**Claim 1** A conjugate for inhibiting the expression of a target gene in a cell, comprising a single nucleic acid strand having a length of at least 25 nucleotides and at most 40 nucleotides covalently bound to one or more ligands, wherein the nucleic acid strand consists of first, second, and third nucleic acid portions, where the first nucleic acid portion is (i) 13 or 14 nucleotides in length and (ii) has a 5'-to-3' directionality, thereby defining a 5' region and a 3' region of the first nucleic acid portion, the second nucleic acid portion is (i) 13 or 14 nucleotides in length and (ii) has a 5'-to-3' directionality, thereby defining a 5' region and a 3' region of the second nucleic acid portion, the first nucleic acid portion and the second nucleic acid portion dimerize to form a blunt-ended, perfectly complementary double-strand, where the first nucleic acid portion is directly linked at its 3' end to the 5' end of the third nucleic acid portion, where the first nucleic acid portion and the third nucleic acid portion are perfectly complementary to a portion of the mRNA transcribed from the target gene, where the third nucleic acid portion is 4 or 5 nucleotides in length and is linked at its 3' end to the 5' end of the second nucleic acid portion, and the one or more ligands comprise one or more N-acetylgalactosamine moieties, conjugate. **Claim 2** The conjugate according to claim 1, wherein the first nucleic acid portion is 14 nucleotides in length. **Claim 3** The conjugate according to claim 1 or 2, wherein the second nucleic acid portion is 14 nucleotides in length. **Claim 4** The conjugate according to any one of claims 1 to 3, wherein the third nucleic acid portion is 5 nucleotides in length. **Claim 5** The conjugate according to any one of claims 1 to 4, comprising 1 to 15 phosphorothioate or phosphorodithioate nucleotide internucleotide linkages, wherein one or more of the phosphorothioate or phosphorodithioate nucleotide internucleotide linkages are in one or more of the 5' region and the 3' region of the first and / or the second nucleic acid portion. **Claim 6** At least one nucleotide of the first nucleic acid portion and / or the second nucleic acid portion and / or the third nucleic acid portion is modified, and at least one of the modifications is a 2'-O-methyl modification or a 2'-F modification in the ribose moiety. The conjugate according to any one of claims 1 to 5.

7. None of the nucleotides at the 2nd or 14th position downstream from the first nucleotide in the 5' region of the first nucleic acid portion contain a 2'-O-methyl modification in the ribose moiety, and / or any nucleotide of the second nucleic acid portion that is positionally corresponding to any nucleotide of the first nucleic acid portion at any position from the 9th to 11th position downstream from the first nucleotide in the 5' region of the first nucleic acid portion does not contain a 2'-O-methyl modification in the ribose moiety. The conjugate according to claim 6.

8. The one or more unmodified nucleotides represent any of the 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, and / or 25th positions downstream from the first nucleotide in the 5' region of the first nucleic acid portion. The conjugate according to any one of claims 1 to 7.

9. The nucleic acid contains at least one vinylphosphonate modification, and the vinylphosphonate modification is in the 5' region of the first nucleic acid portion. The conjugate according to any one of claims 1 to 8.

10. At least one of the one or more nucleotides of the first nucleic acid portion and the second nucleic acid portion is an inverted nucleotide, and is linked to the adjacent nucleotide via the 3'-carbon of the nucleotide and the 3'-carbon of the adjacent nucleotide, and / or is linked to the adjacent nucleotide via the 5'-carbon of the nucleotide and the 5'-carbon of the adjacent nucleotide. The conjugate according to any one of claims 1 to 9.

11. A conjugate for inhibiting the expression of a target gene in a cell, comprising a single nucleic acid strand having a length of at least 25 nucleotides and at most 40 nucleotides covalently bonded to one or more ligands, wherein the nucleic acid strand consists of first, second, and third nucleic acid portions, where The first nucleic acid portion is (i) 13 or 14 nucleotides in length and (ii) has a 5' to 3' directionality, thereby defining a 5' region and a 3' region of the first nucleic acid portion, The second nucleic acid portion is (i) 13 or 14 nucleotides in length and (ii) has a 5' to 3' directionality, thereby defining a 5' region and a 3' region of the second nucleic acid portion, The first nucleic acid portion and the second nucleic acid portion dimerize to form a completely complementary double-strand with blunt ends, wherein the first nucleic acid portion is directly linked at its 3' end to the 5' end of the third nucleic acid portion, wherein the first nucleic acid portion and the third nucleic acid portion are completely complementary to a portion of the mRNA transcribed from the target gene, wherein the third nucleic acid portion is 4 or 5 nucleotides in length and is linked at its 3' end to the 5' end of the second nucleic acid portion, and the one or more ligands comprise one or more N-acetylgalactosamine moieties, and wherein the unpaired nucleotides of the third nucleic acid portion form a bulge structure between the first and the second nucleic acid portions, conjugate.

12. A composition comprising the conjugate according to any one of claims 1 to 11 and a physiologically acceptable excipient.

13. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 11 for use in the treatment of a disease or disorder.

14. The pharmaceutical composition according to claim 13, which is administered subcutaneously or intravenously to an individual in need of treatment.

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