Modified nucleic acid conjugates for inhibiting gene expression

Abstract

The present invention relates to nucleic acid conjugates containing a hydrophobic moiety, such as a 2',3'-O ketal moiety, for inhibiting expression of a target gene.

Description

[Technical Field]

[0001] The present invention relates to nucleic acid conjugates containing a hydrophobic moiety, such as a 2',3'-O ketal moiety, for inhibiting expression of a target gene. [Background technology]

[0002] WO2014 / 048969 describes nucleolipids and methods for preparing nucleolipids. In particular, nucleotides containing a lipid 2',3'-O-ketal moiety are described. These nucleotides have been found to have pharmaceutical activity in certain cancer cells. However, delivery of nucleic acid molecules containing a 2',3'-O-ketal moiety into target cells is not disclosed.

[0003] WO2022 / 144422 discloses nucleic acid conjugates comprising at least one 2',3'-O-ketal moiety and their use for therapy and diagnosis, the contents of which are incorporated herein by reference in their entirety. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide nucleic acid molecules that are highly effective in reducing target gene expression in living organisms. [Means for solving the problem]

[0005] The present inventors have found that siRNA molecules directed against the G12C variant of the KRAS gene molecule, which have a lipid-modified 2',3'-O ketal moiety attached thereto, are highly effective in inhibiting KRAS(G12C) expression or tumor growth in in vivo experimental patient-derived xenograft (PDX) mouse models of colon and lung tumors. Thus, therapeutic nucleic acid conjugates with improved properties are provided. [Brief explanation of the drawings]

[0006] [Figure 1] Structures and molar masses of the 2′,3′-O-ketal moieties PRAMO-01 and PRAMO-02 as phosphoramidites. [Figure 2] Inhibition of KRAS gene expression after administration of lipid-modified siRNA molecules against KRAS(G12C) in PDX peritoneal metastatic colon tumor NMRI nu / nu mice. [Figure 3] Body weight change (A) and tumor volume change (B) after administration of lipid-modified siRNA molecules against KRAS(G12C) in NSCLC PDX Lu7462 NMRI nu / nu mice with heterozygous KRAS G12C. [Figure 4] Time course analysis of tumor growth for all groups (days 0–10 of treatment). Measurement of tumor volume in PDX models (6–8 week-old female NMRI nu / nu mice bearing patient-derived NSCLC tumor Lu7462, 0.04–0.1 cm3 on day 0). Animals were intratumorally administered water for injection (small squares), self-delivered scrambled siRNA (downward-pointing filled triangles), naked siRNA against the KRAS G12C mutation (large squares), or PRAMO self-delivered siRNA against KRAS(G12C) (filled dots). Animals were treated on days 0, 3, 6, and 9. On day 10, animals were sacrificed. Tumors were measured mechanically daily (four mice per group; one mouse from the control group treated with naked siRNA against KRAS(G12C) had to be sacrificed on day 8 of the study due to poor condition and ethical reasons, so data related to this mouse are not included in this graph). Results are shown as the mean ± standard deviation of four independent in vivo experiments. Data were analyzed using one-way ANOVA test. *p≦0.05; **p≦0.01. [Figure 5]Time course analysis of tumor growth for vehicle and treatment groups (days 0–10 of treatment). Measurement of tumor volume in PDX models (6–8-week-old female NMRI nu / nu mice bearing patient-derived NSCLC tumor Lu7462, 0.04–0.1 cm3 on day 0). Animals were intratumorally administered with water for injection (squares) or PRAMO self-delivered siRNA against KRAS(G12C) (black dots). Animals were treated on days 0, 3, 6, and 9. On day 10, animals were sacrificed. Tumors were mechanically measured daily (four mice per group). Results are shown as the mean ± standard deviation of four independent experiments. Data were analyzed using one-way ANOVA. *p ≤ 0.05; **p ≤ 0.01. [Figure 6] Analysis of tumor volume on days 0 and 10 of treatment. Measurement of tumor volume in PDX models (6-8 week-old female NMRI nu / nu mice bearing patient-derived NSCLC tumor Lu7462, 0.04-0.1 cm3 on day 0). Animals were intratumorally administered water for injection (black dots), self-delivered scrambled siRNA (downward-pointing black triangles), naked siRNA against the KRAS G12C mutation (upward-pointing black triangles), or PRAMO self-delivered siRNA against KRAS(G12C) (black squares). Animals were treated on days 0, 3, 6, and 9. Animals were sacrificed on day 10. Tumors were mechanically measured daily (at least four mice per group; one mouse in the control group treated with naked siRNA against KRAS(G12C) had to be sacrificed on day 8 due to poor animal health and ethical reasons). Results are shown as the mean ± standard deviation of four independent experiments. Data were analyzed using one-way analysis of variance. *p≦0.05; **p≦0.01. [Figure 7]Photograph of excised tumors on day 10 of treatment. Comparison of excised tumors from PDX models (6-8 week-old female NMRI nu / nu mice bearing patient-derived NSCLC tumor Lu7462, 0.04-0.1 cm3 on day 0). Animals were intratumorally administered water for injection, PRAMO self-delivered siRNA against KRAS(G12C), self-delivered scrambled siRNA, or naked siRNA against KRAS(G12C). Animals were treated on days 0, 3, 6, and 9. On day 10, animals were sacrificed and tumors were excised. [Figure 8] Western blot analysis of isolated tumors on day 10 of treatment. Western blot analysis of tumors from PDX models (6-8 week-old female NMRI nu / nu mice bearing patient-derived NSCLC tumor Lu7462). Animals were intratumorally administered water for injection, PRAMO self-delivered siRNA against KRAS(G12C), self-delivered scrambled siRNA, or naked siRNA against KRAS(G12C). Animals were treated on days 0, 3, 6, and 9. On day 10, animals were sacrificed and tumors were excised (n=4). DETAILED DESCRIPTION OF THE INVENTION

[0007] A first aspect of the present invention is a nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, wherein the nucleic acid molecule is directed against a target gene and used to inhibit expression of the target gene in a subject.

[0008] In some embodiments, the hydrophobic moiety comprises one, two, or three hydrocarbon groups. In specific embodiments, the hydrophobic moiety comprises two hydrocarbon groups. In some embodiments, the hydrocarbon group comprises at least 5 C atoms, for example, at least 8 C atoms, at least 10 C atoms, at least 12 C atoms, at least 14 C atoms, at least 16 C atoms, or at least 18 C atoms. In some embodiments, at least one hydrocarbon group is a linear hydrocarbon chain. In specific embodiments, all hydrocarbon groups are linear hydrocarbon chains.

[0009] In some embodiments, the nucleic acid molecule of the conjugate is an siRNA molecule. In specific embodiments, the hydrophobic moiety is added to the 5'-end of the sense strand of the siRNA molecule. In more specific embodiments, the hydrophobic moiety is added to the 5'-end of the antisense strand of the siRNA molecule.

[0010] In certain embodiments, the sense and / or antisense strand of the siRNA comprises at least one 3' overhang. In specific embodiments, the overhang has a length of 2 nucleotides and consists of a dT building block.

[0011] In a specific embodiment, the double-stranded portion of the siRNA molecule comprises modified ribonucleotide building blocks, such as 2'-O-methylribonucleotide building blocks and / or 2'-fluororibonucleotide building blocks. In a more specific embodiment, the double-stranded portion of the siRNA molecule consists of modified ribonucleotide building blocks, such as 2'-O-methylribonucleotide building blocks and / or 2'-fluororibonucleotide building blocks.

[0012] In a specific embodiment, 2'-O-methyl and 2'-fluororibonucleotide building blocks are alternately arranged on sense strand and antisense strand.In a more specific embodiment, the double-stranded portion of the siRNA molecule is comprised of the base pair of the first nucleic acid base from 2'-O-methylribonucleotide building block and the second nucleic acid base from 2'-fluororibonucleotide building block, and the first nucleic acid base is complementary to the second nucleic acid base, for example, AU, UA, GC and CG.

[0013] In one embodiment, the target gene is an oncogene. In a specific embodiment, the target gene is a RAS gene, such as a KRAS gene, a HRAS gene, or a NRAS gene.

[0014] RAS genes encode RAS proteins that act as intracellular switches that are turned on by extracellular stimuli, resulting in the transient formation of an active, GTP-bound form of RAS, which activates various signaling pathways that regulate fundamental cellular processes. The mutated RAS oncoprotein is functionally distinct from its normal counterpart: the oncogenic form prevents GAP from increasing the intrinsic catalytic rate of the GTPase, thereby maintaining RAS in a constitutively GTP-bound, active state, which activates oncogenic pathways and cell signaling.

[0015] In some embodiments, the target gene is a wild-type RAS gene, such as a wild-type KRAS, HRAS, or NRAS gene. In these embodiments, the nucleic acid conjugates of the present invention can be used to treat disorders associated with overexpression of the wild-type gene.

[0016] In one embodiment, the target gene is a mutant RAS gene, such as a mutant KRAS, HRAS, or NRAS gene.

[0017] In a specific embodiment, the target gene is a KRAS, HRAS, or NRAS gene mutated at positions 3, 12, 13, 14, 19, 33, 58, 59, 61, 117, 118, and / or 146, wherein the mutation specifically comprises an amino acid substitution. Exemplary mutations in the KRAS gene include, but are not limited to, G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61L, Q61R, A59E, A59G, A59T, K117N, K117R, K117E, A146T, A146P, and A146V. Exemplary mutations in the NRAS gene include, but are not limited to, G12D, G12C, G12S, G13R, G13V, K117R, Q61H, Q61L, Q61K, Q61R, A59D, A59T, and A146T.

[0018] In a more specific embodiment, the target gene is a mutant KRAS gene mutated at position 12 or 13, for example, mutated G12C, G12D, G12V, G12R, G12A, G12S, G13D, and / or G13C.

[0019] In one embodiment, the present invention encompasses the administration of at least two different nucleic acid conjugate molecules, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acid conjugate molecules, directed against different target genes. In a specific embodiment, the at least two different nucleic acid conjugate molecules are directed against different mutant forms of target genes, for example, different mutant forms of the RAS gene. In a more specific embodiment, the at least two different nucleic acid conjugate molecules are directed against different mutant forms of the KRAS gene, for example, G12D, G12V, G12R, and G12A.

[0020] In certain embodiments, the nucleic acid conjugate is used to treat the following types of cancer: - Adrenal cancer, especially adrenocortical tumors - Brain cancer, especially glioblastoma multiforme or low-grade glioma, - Head and neck cancer, especially head and neck squamous cell carcinoma, papillary thyroid carcinoma, or salivary gland tumors; - Endocrine cancer, especially anaplastic or follicular thyroid carcinoma; - Thoracic cancer, especially lung adenocarcinoma (e.g., NSCLC), lung squamous cell carcinoma, thymic carcinoma, or upper aerodigestive tract cancer - Breast cancer, especially invasive breast cancer; - Major gastrointestinal cancer, especially esophageal, gastric, small intestinal, colonic, or rectal adenocarcinoma; - Accessory gastrointestinal cancer, especially hepatocellular carcinoma, gallbladder carcinoma, pancreatic adenocarcinoma, or biliary tract carcinoma; - Genitourinary cancers, especially renal cell carcinoma, bladder urothelial carcinoma, or prostate adenocarcinoma; - gynecological cancers, especially ovarian cancer, endometrial cancer of the uterine corpus, squamous cell carcinoma of the cervix, cervical adenocarcinoma, or endometrial cancer; - Skin cancer, especially cutaneous melanoma; - Hematopoietic / lymphatic cancers, especially acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or plasma cell myeloma; - bone cancer, especially osteosarcoma or malignant fibrous histiocytoma; - Soft tissue cancers, especially soft tissue sarcomas.

[0021] In some embodiments, the nucleic acid conjugate is used to treat colon cancer, for example, colon adenocarcinoma. In specific embodiments, the colon cancer is associated with overexpression of RAS genes, for example, KRAS genes, HRAS genes, or NRAS genes. In more specific embodiments, the colon cancer is associated with mutations of KRAS genes, HRAS genes, or NRAS genes, and may be associated with overexpression. In more specific embodiments, the colon cancer is associated with mutations of KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variants, and may be associated with overexpression.

[0022] In some embodiments, the nucleic acid conjugate is used to treat lung cancer, such as lung adenocarcinoma, particularly non-small cell lung cancer (NSCLC), or lung squamous cell carcinoma. In specific embodiments, the lung cancer is associated with overexpression of RAS genes, such as KRAS, HRAS, or NRAS genes. In more specific embodiments, the lung cancer is associated with mutations, and may be associated with overexpression, of KRAS, HRAS, or NRAS genes. In more specific embodiments, the lung cancer is associated with mutations, and may be associated with overexpression, of KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variants.

[0023] In some embodiments, the nucleic acid conjugate is used for the treatment of brain cancer, particularly glioblastoma multiforme or low-grade glioma.In a specific embodiment, the brain cancer is associated with the overexpression of RAS gene, for example, KRAS gene, HRAS gene, or NRAS gene.In a more specific embodiment, the brain cancer is associated with the mutation of KRAS gene, HRAS gene, or NRAS gene, and may be associated with overexpression.In a more specific embodiment, the brain cancer is associated with the mutation of KRAS gene, HRAS gene, or NRAS gene, and may be associated with overexpression.In a more specific embodiment, the brain cancer is associated with the mutation of KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variant, and may be associated with overexpression.

[0024] In some embodiments, the nucleic acid conjugate is used for the treatment of pancreatic cancer, particularly pancreatic adenocarcinoma.In a specific embodiment, the pancreatic cancer is associated with the overexpression of RAS gene, such as KRAS gene, HRAS gene, or NRAS gene.In a more specific embodiment, the pancreatic cancer is associated with the mutation of KRAS gene, HRAS gene, or NRAS gene, and may be associated with overexpression.In a more specific embodiment, the pancreatic cancer is associated with the mutation of KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variant, and may be associated with overexpression.

[0025] In some embodiments, the nucleic acid conjugate is used for treating gynecological cancer, particularly ovarian cancer.In a specific embodiment, the gynecological cancer is associated with overexpression of RAS gene, such as KRAS gene, HRAS gene, or NRAS gene.In a more specific embodiment, the gynecological cancer is associated with mutation of KRAS gene, HRAS gene, or NRAS gene, and may be associated with overexpression.In a more specific embodiment, the gynecological cancer is associated with mutation of KRAS gene, HRAS gene, or NRAS gene, and may be associated with overexpression.In a more specific embodiment, the gynecological cancer is associated with mutation of KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variant, and may be associated with overexpression.

[0026] In some embodiments, the nucleic acid conjugate is used for the treatment of skin cancer, particularly cutaneous melanoma. In specific embodiments, the skin cancer is associated with overexpression of RAS genes, such as KRAS, HRAS, or NRAS genes. In more specific embodiments, the skin cancer is associated with mutations in KRAS, HRAS, or NRAS genes, and may be associated with overexpression. In more specific embodiments, the skin cancer is associated with mutations in KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variants, and may be associated with overexpression.

[0027] In some embodiments, administration of the nucleic acid conjugate, e.g., single or multiple administrations, inhibits expression of the target gene by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a control.

[0028] In certain embodiments, the subject is a mammalian subject, particularly a human subject.

[0029] In a further embodiment of the invention, the nucleic acid conjugate is a nucleic acid conjugate of formula (I) comprising at least one 2',3'-O-ketal moiety: JPEG2026503773000001.jpg55153where, X is a nucleic acid molecule directed against a target gene, B is a nucleobase, and R1 and R2 are each independently a hydrocarbon group, which may contain one or more heteroatoms such as N, O, P, S, or halo.

[0030] A further aspect of the present invention relates to a method for inhibiting expression of a target gene in a subject, said method comprising the step of administering to said subject a therapeutically effective amount of a nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, in particular a nucleic acid conjugate of formula (I) comprising at least one 2',3'-O-ketal moiety: JPEG2026503773000002.jpg50153where, X is a nucleic acid molecule directed against the target gene, B is a nucleobase, and R1 and R2 are each independently a hydrocarbon group, which may contain one or more heteroatoms such as N, O, P, S, or halo.

[0031] It is understood that the present invention also includes any stereoisomers, salts, and solvates of the compounds depicted in formula (I) and any other formula herein.

[0032] The nucleic acid conjugate of formula (I) comprises a nucleic acid molecule, X, having attached thereto at least one 2',3'-O-ketal moiety.

[0033] The term "nucleic acid molecule" refers to any type of oligonucleotide or polynucleotide that contains at least two nucleotide building blocks. A nucleic acid molecule can contain ribonucleotide building blocks, 2'-deoxyribonucleotide building blocks, modified nucleotide building blocks, or any combination thereof. In certain embodiments, the nucleic acid molecule is an RNA molecule that may contain at least one modified nucleotide building block and / or at least one 2'-deoxyribonucleotide building block, a DNA molecule that may contain at least one modified nucleotide building block and / or at least one ribonucleotide building block, or a nucleic acid analog molecule composed of modified nucleotide building blocks.

[0034] As used herein, the term "nucleic acid analog molecule," specifically a DNA or RNA molecule analog, refers to a molecule that is similar to, i.e., structurally similar to, a naturally occurring RNA or DNA molecule. Generally, naturally occurring nucleic acids are chains of nucleotides, which are composed of three elements: a backbone portion (specifically, a phosphate backbone), a pentose (either ribose or deoxyribose), and one of four nucleic acid bases. Analogs may have any of these elements altered, such as modified nucleic acid bases or locked nucleic acids (LNA). Corresponding analogs are well known to those skilled in the art. Analogs may, among other things, impart different base-pairing and base-stacking properties.

[0035] The 2',3'-O ketal moiety comprises a nucleobase B. The term "nucleobase" generally refers to a cyclic (e.g., monocyclic or bicyclic), saturated, unsaturated, aromatic, or heteroaromatic base containing at least one N atom, wherein the nucleobase can form a base pair with a complementary nucleobase, particularly a naturally occurring nucleobase, via hydrogen bonding. The nucleobase may be any naturally occurring or unnatural nucleobase, for example, any naturally occurring or unnatural purine or pyrimidine base, such as adenine, cytosine, guanine, thymine, or uracil, or a modified nucleobase, for example, a modified adenine, cytosine, guanine, thymine, or uracil base.

[0036] The nucleobase of the 2',3'-O ketal moiety can be a "modified nucleobase." This term includes any type of modified nucleobase, e.g., a nucleobase substituted with a hydrophobic moiety, a nucleobase substituted with a carbohydrate moiety, and / or a nucleobase substituted with a functional moiety.

[0037] The hydrophobic moiety may be selected from moieties having at least 5 C atoms, in particular at least 10 C atoms.

[0038] In some embodiments, the hydrophobic moiety is selected from a substituted or unsubstituted acyclic or cyclic terpene moiety. For example, the hydrophobic moiety is a C5-C 30 The terpene portion, specifically C5-C 20 The terpene portion, more specifically C 15 They may be terpene moieties, which may be cyclic or acyclic, saturated or unsaturated, and / or optionally substituted or interrupted with heteroatom(s) or functional group(s), or may be sterol moieties, e.g., cholesterol moieties.

[0039] Additionally, the hydrophobic moiety may be selected from substituted or unsubstituted, cyclic or acyclic, saturated, unsaturated, or polyunsaturated carboxylic acid moieties, including salts or derivatives thereof, such as esters or amides. For example, the hydrophobic moiety may be selected from C5-C 30 It may be a carboxylic acid moiety, such as a docosahexaenoic acid moiety, an eicosapentaenoic acid moiety, a docosanoic acid moiety, a lithocholic acid moiety, or a retinoic acid or retinoic acid ester moiety.

[0040] Additionally, the hydrophobic moiety may be selected from substituted or unsubstituted, cyclic or acyclic, saturated, unsaturated, or polyunsaturated alcohols, ketones, aldehydes, or amines. For example, the hydrophobic moiety may be a C5-C 30 It may be an alcohol, ketone, or amide moiety, such as a tocopherol moiety, a tocopherol succinate moiety, a retinol moiety, a retinal moiety, a spermine moiety, or a spermidine moiety.

[0041] In certain embodiments, the hydrophobic moiety is: JPEG2026503773000003.jpg22153 and / or Selected from JPEG2026503773000004.jpg32153, where R and R′ are independently C1-C 30 Alkyl, preferably C5-C 25 alkyl, n is an integer ranging from 1 to 6, preferably n is 1 or 2, and a is an integer ranging from 1 to 20, preferably from 2 to 18, more preferably from 6 to 16.

[0042] The carbohydrate moiety may be a monosaccharide, oligosaccharide, or polysaccharide moiety, including modified carbohydrates, such as acetylated carbohydrate moieties. For example, the carbohydrate moiety may be a GalNAc moiety.

[0043] The functional moiety may be a click functional moiety such as an alkyne or azide group, or may be an ether, ester, amide, carboxylic acid, thioester, thioamide, or thioether group.

[0044] The conjugate of formula (I) comprises a 2',3'-O-ketal moiety in which the 2'-OH and 3'-OH groups of the ribose moiety are converted to a ketal group containing substituents R1 and R2.

[0045] R1 and R2 are, independently of each other, a hydrocarbon group, which may contain one or more heteroatoms, such as N, O, P, S, or halo.

[0046] R1 and R2 may be saturated, monovalent or polyunsaturated, acyclic, straight-chain or branched hydrocarbon groups, or saturated, monovalent or polyunsaturated, aromatic or heteroaromatic cyclic hydrocarbon groups, which may be interrupted or substituted by one or more heteroatoms.

[0047] In one embodiment, R1 and / or R2 are independently selected from the group consisting of C 5-21 Hydrocarbon groups, specifically C 5-11 A hydrocarbon group, more specifically a C hydrocarbon group, which may contain one or more heteroatoms such as N, O, P, S, or halo (e.g., F, Cl, Br, or I).

[0048] In further embodiments, R1 and / or R2 are, independently of each other, a straight-chain or branched alkyl group, a straight-chain or branched alkenyl group, and a straight-chain or branched alkynyl group, wherein the alkyl, alkenyl, or alkynyl group may contain one or more heteroatoms, such as N, O, P, S, or halo.

[0049] In still further embodiments, R1 and / or R2 are, independently of each other, cyclic moieties, i.e., moieties containing at least one ring structure (e.g., a monocyclic, bicyclic, or tricyclic structure). The cyclic moiety may be a carbocyclic or heterocyclic moiety (e.g., 5 to 18 ring atoms), in which at least one C atom may be replaced by at least one heteroatom selected from N, O, and S.

[0050] In still further embodiments, R1 and / or R2, independently of each other, comprise a functional group selected from an ether group, an ester group, an amide group, a carboxylic acid group, a thioester group, a thioamide group, or a thioether group.

[0051] In still further embodiments, R1 and / or R2, independently of each other, comprise a marker group, such as a fluorescent, luminescent, or radioactive marker group.

[0052] In still further embodiments, R1 and / or R2, independently of each other, comprise a nucleic acid molecule as defined herein, for example a nucleic acid molecule having up to 50, particularly 5 to 25, nucleotide building blocks.

[0053] In specific embodiments, R1 and R2 are straight-chain or branched C9 alkyl groups, more specifically straight-chain C9 alkyl groups.

[0054] In a specific embodiment, the conjugate comprises a nucleobase B that is a uracil base of formula (IIa): JPEG2026503773000005.jpg47153 where Z is CH or N, and R3 is H.

[0055] In this embodiment, R1 and R2 are preferably straight chain C9 alkyl groups.

[0056] In a further specific embodiment, the conjugate comprises a nucleobase B that is a uracil base of formula (IIb): JPEG2026503773000006.jpg60153 where Z is CH or N, and R3 is a hydrophobic moiety, such as a terpene moiety, specifically C5-C 30 The terpene portion, more specifically C5-C 20 The terpene portion, more specifically C 15 A terpene moiety, for example a terpene moiety of formula (III): JPEG2026503773000007.jpg30153

[0057] In this embodiment, R1 and R2 are preferably straight chain C9 alkyl groups.

[0058] In still further specific embodiments, the 2',3'-O-ketal moiety appended to the conjugate is derived from compound PRAMO-01 or compound PRAMO-02 shown in FIG.

[0059] In certain embodiments, the nucleic acid molecule (e.g., nucleic acid molecule X) of the nucleic acid conjugate described herein above has a chain length of at least about 5 nucleotide building blocks up to 1000 or more nucleotide building blocks. In further embodiments, the nucleic acid molecule has a chain length of between about 5 and about 100 nucleotide building blocks, between about 10 and about 50 nucleotide building blocks, between about 12 and about 40 nucleotide building blocks, specifically between about 15 and about 30 nucleotide building blocks.

[0060] The term "nucleic acid molecule" encompasses single-, double-, triple-, and quadruple-stranded nucleic acid molecules, such as DNA or RNA molecules, and analogs thereof. Additionally, nucleic acid molecules may contain at least one modified nucleotide building block, as described below.

[0061] In one embodiment, the nucleic acid molecule is a DNA molecule, and may comprise at least one modified nucleotide building block.The term "DNA molecule" includes single-stranded and multi-stranded, such as double-stranded, triple-stranded, or quadruple-stranded DNA molecules, specifically single-stranded or double-stranded DNA molecules.Multi-stranded, such as double-stranded DNA molecules, may comprise strands of the same length or strands of different lengths.In multi-stranded, such as double-stranded DNA molecules, each strand may exist as a separate molecule, or may be covalently linked via single-stranded loops or heterologous linkers.

[0062] The term "DNA molecule" encompasses molecules composed of naturally occurring DNA building blocks, i.e., 2'-deoxyribonucleotide building blocks, as well as molecules that contain at least one 2'-deoxyribonucleotide building block and at least one modified nucleotide building block, and / or at least one ribonucleotide building block.

[0063] In a further embodiment, the nucleic acid molecule is an RNA molecule and may contain at least one modified building block and / or at least one 2'-deoxyribonucleoside building block. The term "RNA molecule" encompasses single-stranded or multi-stranded, such as double-stranded, triple-stranded, and quadruple-stranded RNA molecules, specifically single-stranded or double-stranded RNA molecules. Multi-stranded RNA molecules may contain strands of the same length or different lengths. For example, double-stranded RNA molecules may be blunt-ended or may have at least one overhang, such as at least one 3'-overhang. In multi-stranded, e.g., double-stranded RNA molecules, the individual strands may exist as separate molecules or may be covalently linked via single-stranded loops or heterologous linkers.

[0064] The term "RNA molecule" includes molecules composed of naturally occurring RNA building blocks, i.e., ribonucleotide building blocks, and molecules that contain naturally occurring RNA building blocks and at least one modified nucleotide building block, and / or at least one 2'-deoxyribonucleoside building block.

[0065] The term "nucleotide building block" refers to a moiety that can be incorporated into a nucleic acid molecule described herein to form part of said nucleic acid molecule. Typically, a nucleotide building block comprises a nucleobase moiety, a sugar moiety, and a backbone moiety. The term "nucleobase" can encompass any type of nucleobase, as described herein above for nucleobase B of formula (I). The sugar moiety can comprise a ribose moiety or a 2'-deoxyribose moiety, including any modifications thereof. The backbone moiety can comprise a phosphoester group that forms an internucleoside linkage between two nucleotide building blocks, including any modifications thereof. Nucleic acid molecule X can comprise modified nucleotide analog blocks, including nucleobase modifications, sugar modifications, and / or backbone modifications.

[0066] In certain embodiments, the nucleic acid molecule comprises at least one modified nucleotide building block, specifically selected from: (a) Nucleobase modified building blocks; (b) glycosylated building blocks; (c) backbone-modified building blocks, and (d) any combination thereof.

[0067] The term "nucleobase-modified building block" refers to a nucleotide building block that comprises a modified nucleobase. The term "modified nucleobase" can encompass any type of modified nucleobase, as described herein above for nucleobase B of Formula (I).

[0068] The term "sugar-modified building block" refers to a nucleotide building block that includes a modified sugar. The term "sugar" typically refers to a ribose or 2'-deoxyribose moiety and includes any type of modified sugar moiety. In certain embodiments, sugar-modified nucleotide building blocks are selected from nucleotide building blocks in which the ring and / or substituents on the ring of the ribose moiety are modified.

[0069] In certain embodiments, the modified nucleotide building block is a 2'-modified nucleotide building block in which the 2'-OH substituent of the ribose moiety is replaced with 2'-R4, where R4 is halo (e.g., fluoro), C1-C5 alkyl, O-C1-C5 alkyl, S-C1-C5 alkyl, C2-C5 alkenyl, O-C2-C5 alkenyl, S-C2-C5 alkenyl, C2-C5 alkynyl, O-C2-C5 alkynyl, S-C2-C5 alkynyl, amino, including mono- or di-substituted amino (e.g., C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl-substituted amino), where each alkyl, alkenyl, or alkynyl group may be substituted with OH, halo, cycloalkyl, cycloalkenyl, (hetero)aryl (e.g., phenyl), O-alkyl, S-alkyl, and / or amino.

[0070] In certain embodiments, the modified nucleotide building block is a bridged (e.g., 2'-4' bridged) modified nucleotide building block, where the bridge is typically 2-5 atoms, specifically 2-3 atoms in length, and includes a C atom and may include a heteroatom such as O, N, or S. In a specific embodiment, the modified building block is a locked nucleic acid building block having a 2'-O-CH2-4' bridge.

[0071] In certain embodiments, a modified nucleotide building block is a building block in which the ring of the ribose moiety is modified, such as a morpholino building block, a thioribose building block, a six-membered pyranose building block, or a peptide nucleic acid (PNA) building block.

[0072] The term "backbone-modified building block" refers to a nucleotide building block that includes a modified internucleoside linkage in which the phosphoester groups connecting adjacent building blocks are replaced by modified internucleoside linkages, such as phosphorothioate linkages, alkylphosphonate (e.g., methylphosphonate) linkages, and boranophosphate linkages.

[0073] The nucleic acid molecule is preferably selected from nucleic acid molecules suitable for pharmaceutical use.Preferred nucleic acid molecules include: mRNA molecules containing coding sequences, particularly mRNA molecules containing protein-coding sequences; RNA molecules capable of RNA interference, such as 16-27-mer siRNA, particularly 21-mer siRNA, blunt siRNA, sisiRNA, shRNA, asiRNA, aiRNA, Fork siRNA, 27-mer siRNA, Dumbbell siRNA, 16-mer siRNA, ss-siRNA; microRNA molecules and their antagomirs, such as pre-miRNA mimics; RNA molecules capable of gene editing, such as components of CRISPR / Cas complexes, such as tracrRNA, crRNA, sgRNA; antisense DNA or RNA; nucleic acid molecules that form triplexes or quadruplexes; CpG-oligonucleotides, TTAGGG-oligonucleotides, aptamers, ribozymes, or DNAzymes, or precursors or modified versions of such molecules.

[0074] In certain embodiments, the nucleic acid molecule is an RNA molecule which may comprise at least one modified building block and / or at least one DNA building block, a DNA molecule which may comprise at least one modified building block and / or at least one RNA building block, or a nucleic acid analog molecule composed of modified building blocks.

[0075] In a further embodiment, the nucleic acid molecule is a double-stranded RNA molecule, which may include at least one modified building block and may have at least one overhang (e.g., a 5'-overhang and / or a 3'-overhang, e.g., an overhang of 1, 2, or 3 nucleotide building blocks). In a specific embodiment, the RNA molecule is an siRNA molecule.

[0076] In a further embodiment, the nucleic acid molecule is a single-stranded DNA molecule, in particular an antisense molecule, which may comprise at least one modified building block and / or at least one ribose building block.

[0077] Nucleic acid conjugates (e.g., nucleic acid conjugates of formula (I)) may be prepared by adding a hydrophobic moiety (e.g., a 2'-3'-O-ketal moiety (IV)) to a nucleic acid molecule, where the 2'-3'-O-ketal moiety is of formula (IV): JPEG2026503773000008.jpg54153where, Y is a reactive functional moiety, such as a phosphoramidate group, or another group suitable for chemical synthesis of nucleic acid molecules (particularly nucleic acid molecule backbones); B is a nucleobase, and R1 and R2 are, independently of each other, a hydrocarbon group, which may contain one or more heteroatoms, such as N, O, P, S, or halo.

[0078] In still further specific embodiments, the 2',3'-O-ketal moiety (IV) is compound PRAMO-01 or compound PRAMO-02 shown in FIG.

[0079] As used herein, the term "phosphoramidate" can also include "phosphoramidate variants" that are used and known to those skilled in the art for the synthesis of nucleic acid molecules.

[0080] The synthesis of the 2',3'-O-ketal moiety (IV) is described in WO2014 / 048969, the disclosure of which is incorporated herein by reference. The addition of the 2',3'-O-ketal moiety (IV) or any other hydrophobic moiety to a nucleic acid molecule can be carried out during or after the synthesis of the nucleic acid molecule, for example, according to standard methods in solid-phase nucleic acid synthesis.

[0081] In certain embodiments, a hydrophobic moiety, such as a 2',3'-O-ketal building block, is added to the 5' end and / or the 3' end of at least one strand of the nucleic acid molecule.

[0082] In a specific embodiment, a hydrophobic moiety, such as a 2',3'-O-ketal building block, is added to the 5' end of the sense strand of the double-stranded siRNA molecule or to the 5' end of the antisense molecule.

[0083] In a further embodiment, hydrophobic moieties, such as 2',3'-O-ketal building blocks, are attached to nucleobases within the strand of a nucleic acid molecule.

[0084] In a further embodiment, the hydrophobic moieties, eg, 2',3'-O-ketal building blocks, added to the nucleic acid molecule are multiple, eg, two.

[0085] A further aspect of the present invention relates to the use of the nucleic acid conjugates described herein, e.g., the conjugates of Formula (I), in medicine, e.g., for use in human or veterinary medicine, particularly for use in human medicine and in vivo animal research. In certain embodiments, the pharmaceutical use involves administering the conjugate to a subject in need thereof, particularly a human subject. In a further embodiment, the conjugate is administered ex vivo to a target cell or target organ, which is then introduced into a subject in need thereof, particularly a human subject. In yet a further embodiment, the conjugate is administered to an oocyte or embryo, excluding the use of human stem cells or human embryos for industrial commercial purposes. When the conjugates of the present invention are administered to an oocyte or embryo, it is preferred that the germline of the oocyte or embryo is not genetically modified, and only a disease, such as a genetic disease, is treated. According to a preferred embodiment, the conjugates of the present invention are administered to a non-human oocyte or non-human embryo.

[0086] The present invention provides an efficient method for mediating target-specific nucleic acid modification in a cell, tissue, organ, or organism, comprising the steps of: (a) contacting a cell, tissue, organ, or organism with a conjugate of the invention; and (b) mediating a target-specific nucleic acid modification to the target nucleic acid caused by the nucleoside component of the conjugate.

[0087] The contacting step (a) may comprise introducing the conjugate into a target cell (e.g., an isolated target cell), which may be present in a cell culture, a unicellular microorganism, or a target cell or cells (e.g., a target tissue and / or a target organ) within a multicellular organism. The target cell, target tissue, and / or target organ is preferably a mammal, including a human. The target organism is preferably a mammalian organism, such as a human organism. The target cell is preferably a mammalian cell, including a human cell. The target tissue is preferably a mammalian tissue, including a human tissue. The target organ is preferably a mammalian organ, including a human organ.

[0088] Introduction into an organism can include any type of administration, including systemic or local administration, for example, administration by injection or infusion. Exemplary types of administration include: auricular, buccal, intrabronchial, enteral, epidural, inhalation, instillation into the bladder, intra-articular, intragastric, intragluteal, intracardiac, intradermal, intralumbar, intralymphatic, intramammary, intramuscular, intranasal, intraneuronal, intraocular, intraosseous, intraperitoneal, intrapleural, intrapulmonary, intraluminal, intrathecal, intratracheal, intraurethral, ​​intrauterine, intravenous, intraventricular, intravitreal, oral, peroral, parenteral, epidural, perineural, percutaneous, rectal, retrobulbar, subconjunctival, subcutaneous, sublingual, topical, transdermal, transmucosal, and / or intravaginal administration.

[0089] The mediating step (b) preferably involves modification of the target nucleic acid, for example by RNA interference when using an siRNA conjugate, or by inhibition of mRNA transcription when using an antisense molecule conjugate.

[0090] The present invention also provides a pharmaceutical composition comprising the above-mentioned conjugate as an active agent together with a suitable carrier.For diagnostic or therapeutic use, the pharmaceutical composition can be in the form of a solution (for example, a solution for infusion or injection), a cream, an ointment, a tablet, a suspension, a powder, etc.The composition can be administered in any suitable form, for example, by parenteral administration (for example, injection or infusion), by transmucosal application, by transdermal application, or by oral, topical, nasal, rectal application, etc.Topical application is particularly preferred, for example, intraperitoneal or intraperitoneal during surgery.More preferably, intratumoral or intraocular application is applied near the tumor, particularly for the siRNA conjugate of the present invention.

[0091] The pharmaceutical composition may comprise the conjugate as an active agent or prodrug in any form suitable for delivery into an organism, particularly into a human organism. For example, the composition may comprise the active agent in encapsulated or non-encapsulated form, with a delivery vehicle such as a liposome, and / or with a transfection reagent, or without a delivery vehicle and / or without a transfection reagent.

[0092] In certain embodiments, the conjugates of the present invention may be used in the modulation, e.g., down-regulation or up-regulation, of a gene of interest in a target cell, tissue, organ, or organism. The gene of interest may be an endogenous gene or a gene from an exogenous pathogen, in particular a viral or bacterial gene, or an endogenous disease-related gene, such as an oncogene or an autoimmune or allergic disease-related gene.

[0093] In a further embodiment, the conjugates of the invention may be used in the transfer and optionally expression of a coding (eg protein-coding) nucleic acid molecule, such as an mRNA molecule, in a target cell or target organism.

[0094] A specific aspect of the present invention relates to the use of conjugate for specifically delivering nucleic acid molecules, particularly therapeutic nucleic acid molecules as described herein, to target cells, target tissues, and / or target organs.A particularly preferred embodiment of the present invention relates to the use of hydrophobic moiety (for example, 2',3'-O-ketal building block as described herein), which is added to the 5' end of the sense strand of double-stranded siRNA molecules or to the 5' end of antisense molecules, for specifically delivering target cells, target tissues, and / or target organs, particularly for in vivo delivery.Therefore, it is particularly preferred to use siRNA conjugate for specifically delivering siRNA molecules, particularly therapeutic siRNA molecules, to target cells, target tissues, and / or target organs.

[0095] For example, the target cell can be a lung cell, heart cell, kidney cell, liver cell, pancreatic cell, colon cell, muscle cell, nerve cell, stomach cell, small intestine cell, large intestine cell, rectum cell, bladder cell, bone cell, adrenal gland cell, eye cell, skin cell, or brain cell. As used herein, "tissue" refers to an organism made up of homogeneous or heterogeneous groups of differentiated cells, such as target cells, that have a common function or structure.

[0096] A still further aspect of the present invention relates to the in vitro use of the conjugate to deliver a nucleic acid molecule into a target cell, tissue, or organ. The target cell can be selected from any type of cell to which a nucleic acid molecule can be delivered, such as an animal cell (e.g., a mammalian cell, an avian cell, or an insect cell), a plant cell, a fungal cell, a protist cell, a bacterial cell, and an archaeal cell. In a specific embodiment, the target cell is a human cell.

[0097] A specific embodiment of this aspect relates to the use of the conjugate in down-regulating genes in target cells, tissues, and / or organs.

[0098] Specifically, the present invention provides target cell-specific, target tissue-specific, and / or target organ-specific delivery of nucleic acid molecules in vitro. For example, the target cell can be a lung cell, a heart cell, a kidney cell, a liver cell, a pancreatic cell, a colon cell, a muscle cell, a nerve cell, a stomach cell, a small intestine cell, a large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, an eye cell, a skin cell, or a brain cell.

[0099] A still further aspect of the present invention relates to the use of the 2',3'-O-ketal moiety (IV) described herein for attachment to nucleic acid molecules.

[0100] Further aspects of the present invention are as described in the following sections of this specification.

[0101] [item] 1. A nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, the nucleic acid molecule is directed against a target gene; used to inhibit expression of the target gene in a subject. Nucleic acid conjugates.

[0102] 2. A nucleic acid conjugate of formula (I) comprising at least one 2',3'-O-ketal moiety: JPEG2026503773000009.jpg58153where, X is a nucleic acid molecule directed against a target gene, B is a nucleobase, and R1 and R2 are each independently a hydrocarbon group; The hydrocarbon group may contain one or more heteroatoms, such as N, O, P, S, or halo; used to inhibit expression of the target gene in an organism, Nucleic acid conjugates.

[0103] 3. The conjugate according to item 2, where R1 and R2 are independently C 5-21 Hydrocarbon groups, specifically C 5-11 a hydrocarbon group, more specifically a C9 hydrocarbon group, The hydrocarbon group may contain one or more heteroatoms, such as N, O, P, S, or halo. Conjugates.

[0104] 4. The conjugate according to item 2 or 3, wherein R1 and R2 are, independently of each other, a linear or branched alkyl group, a linear or branched alkenyl group, and a linear or branched alkynyl group; The alkyl, alkenyl, or alkynyl groups may contain one or more heteroatoms, such as N, O, P, S, or halo; Conjugates.

[0105] 5. The conjugate according to any one of items 2 to 4, wherein R1 and R2 are linear or branched C9 alkyl groups; Conjugates.

[0106] 6. The conjugate according to any one of items 2 to 5, The nucleobase B is a monocyclic or bicyclic aromatic or heteroaromatic base containing at least one N atom, Conjugates.

[0107] 7. The conjugate according to any one of items 2 to 6, The nucleobase B is a heterocyclic base, such as a purine or pyrimidine base, specifically selected from uracil, thymine, or analogs thereof; Conjugates.

[0108] 8. The conjugate according to any one of items 2 to 7, the nucleobase B is a modified base, e.g., a base substituted with a hydrophobic moiety, a base substituted with a carbohydrate moiety, or a base substituted with a functional moiety; Conjugates.

[0109] 9. The conjugate according to item 8, The hydrophobic portion is a terpene portion, specifically C5-C 30 The terpene portion, more specifically C5-C 20 The terpene portion, more specifically C 15 a terpene moiety, or a sterol moiety, such as a cholesterol moiety; Conjugates.

[0110] 10. The conjugate according to item 8, the carbohydrate moiety is a GalNAc group. Conjugates.

[0111] 11. The conjugate according to item 8, the functional moiety is a click functional moiety such as an alkyne or azide group, or an alkyne or azide group, or an ether group, ester group, amide group, carboxylic acid group, thioester group, thioamide group, or thioether group, Conjugates.

[0112] 12. The conjugate according to any one of items 2 to 7, wherein B is a nucleobase of formula (IIa): JPEG2026503773000010.jpg62153 where Z is C-H or N, and R3 is H; Conjugates.

[0113] 13. The conjugate according to any one of items 2 to 9, wherein B is a nucleobase of formula (IIb): JPEG2026503773000011.jpg60153 where Z is CH or N, and R3 is a terpene moiety, specifically C5-C 30 The terpene portion, more specifically C5-C 20 The terpene portion, more specifically C 15 A terpene moiety, for example a terpene moiety of formula (III): JPEG2026503773000012.jpg27153 conjugate.

[0114] 14. The conjugate of any one of the preceding paragraphs, The nucleic acid molecule has a chain length of at least 5 nucleotide building blocks to 1000 or more nucleotide building blocks, between 5 and 100 nucleotide building blocks, between 10 and 50 nucleotide building blocks, between 12 and 40 nucleotide building blocks, and particularly between 15 and 30 nucleotide building blocks. Conjugates.

[0115] 15. The conjugate of any one of the preceding paragraphs, The nucleic acid molecule comprises at least one modified nucleotide building block, wherein the modified nucleotide building block is specifically: (a) Base-modified nucleotide building blocks; (b) sugar-modified nucleotide building blocks; (c) backbone-modified nucleotide building blocks; and (d) any combination thereof; Selected from: Conjugates.

[0116] 16. The conjugate according to item 15, the base-modified nucleotide building blocks are selected from building blocks comprising a base substituted with a hydrophobic moiety, nucleotide building blocks comprising a base substituted with a carbohydrate moiety, and nucleotide building blocks comprising a base substituted with a functional moiety; Conjugates.

[0117] 17. The conjugate according to item 15, The sugar-modified nucleotide building blocks are selected from 2'-modified ribose building blocks, 2'-4' bridged modified ribose building blocks, such as locked nucleotide (LNA) building blocks, morpholino building blocks, and peptide nucleic acid (PNA) building blocks; Conjugates.

[0118] 18. The conjugate according to item 15, The backbone-modified nucleotide building blocks include modified internucleoside linkages, such as phosphorothioate linkages, alkylphosphonate (e.g., methylphosphonate) linkages, and boranophosphate linkages. Conjugates.

[0119] 19. The conjugate of any one of the preceding items, The nucleic acid molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. Conjugates.

[0120] 20. The conjugate according to any one of items 1 to 19, The nucleic acid molecule (i) an RNA molecule, which may comprise at least one modified nucleotide building block and / or at least one DNA building block; or (ii) a DNA molecule, which may comprise at least one modified nucleotide building block and / or at least one RNA building block; or (iii) nucleic acid analog molecule That is, Conjugates.

[0121] 21. The conjugate according to item 20, the nucleic acid molecule is a single-stranded, double-stranded, triple-stranded, or quadruple-stranded RNA molecule, particularly a single-stranded or double-stranded RNA molecule; The RNA molecule may comprise at least one deoxyribonucleotide building block, at least one modified building block, and may have at least one 3'-overhang, in particular an siRNA molecule. Conjugates.

[0122] 22. The conjugate according to item 21, the nucleic acid molecule is a single-stranded, double-stranded, triple-stranded, or quadruple-stranded RNA molecule, in particular a single-stranded or double-stranded DNA molecule; The DNA molecule may comprise at least one ribonucleotide building block, at least one modified building block, in particular an antisense molecule, Conjugates.

[0123] 23. A conjugate according to any one of the preceding paragraphs, The hydrophobic moiety, e.g., a 2',3'-O-ketal building block, is added to the 5' end and / or the 3' end of the strand of the nucleic acid molecule. Conjugates.

[0124] 24. The conjugate according to item 23, The hydrophobic moiety, e.g., the 2',3'-O-ketal building block, is added to the 5' end of the sense strand of the double-stranded siRNA molecule or to the 5' end of the antisense strand. Conjugates.

[0125] 25. A conjugate according to any one of the preceding paragraphs, the hydrophobic moiety, e.g., the 2',3'-O-ketal building block, is attached to a base within the strand of the nucleic acid molecule; Conjugates.

[0126] 26. A conjugate according to any one of the preceding paragraphs, for use in medicine, for example for use in human or veterinary medicine, in particular for use in human medicine, Conjugates.

[0127] 27. A conjugate according to any one of items 1 to 25 for the use according to item 26, The conjugate is administered to a subject in need thereof, particularly a human subject. Conjugates.

[0128] 28. A conjugate according to any one of items 1 to 25 for the use according to item 26, The conjugate is administered to a target cell or target organ ex vivo, and the target cell or target organ is then introduced into a subject in need thereof, particularly a human subject. Conjugates.

[0129] 29. A conjugate according to any one of items 1 to 25 for the use according to item 26, the conjugate is administered to an oocyte or embryo; The use of human stem cells or human embryos for industrial commercial purposes is excluded here. Conjugates.

[0130] 30. A conjugate according to any one of items 1 to 25 for the use according to any one of items 26 to 29, The administration includes target cell and / or target organ specific delivery. Conjugates.

[0131] 31. A conjugate according to any one of items 1 to 25 for the use according to any one of items 26 to 30, for use in the up-regulation or down-regulation of genes in target cells or target organisms, for example endogenous genes or genes from exogenous pathogens, in particular viral or bacterial genes, or endogenous disease-associated genes such as cancer genes or genes associated with autoimmune or allergic diseases, Conjugates.

[0132] 32. A conjugate according to any one of items 1 to 25 for the use according to any one of items 26 to 30, for use in the transfer and, optionally, expression of an encoding nucleic acid molecule gene in a target cell or target organism, Conjugates.

[0133] 33. A conjugate according to any one of items 1 to 25 for the use according to any one of items 26 to 32, the target cell is a lung cell, a heart cell, a kidney cell, a liver cell, a pancreatic cell, a colon cell, a muscle cell, a nerve cell, a stomach cell, a small intestine cell, a large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, an eye cell, a skin cell, or a brain cell; Conjugates.

[0134] 34. Use of a conjugate according to any one of items 1 to 25 in vitro for delivering a nucleic acid molecule into a target cell.

[0135] 35. The use according to item 34, The target cell is selected from an animal cell, such as a mammalian cell, an avian cell, or an insect cell, a plant cell, a fungal cell, a protist cell, a bacterial cell, and an archaeal cell; use.

[0136] 36. The use according to item 34 or 35, the target cell is a human cell; use.

[0137] 37. The use according to any one of items 34 to 36, For upregulation or downregulation of genes in the target cells, use.

[0138] 38. The use according to any one of items 34 to 36, for the introduction and, optionally, expression of an encoding nucleic acid molecule gene in a target cell or target organism, use.

[0139] 39. The use according to any one of items 34 to 38, including target cell-specific delivery; use.

[0140] 40. The use according to any one of items 34 to 39, the target cell is a lung cell, a heart cell, a kidney cell, a liver cell, a pancreatic cell, a colon cell, a muscle cell, a nerve cell, a stomach cell, a small intestine cell, a large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, an eye cell, a skin cell, or a brain cell; use.

[0141] 41. Use of a 2′,3′-O-ketal moiety (IV): JPEG2026503773000013.jpg50153where, Y is a reactive functional moiety; B is a nucleobase, and R1 and R2 are each independently a hydrocarbon group; The hydrocarbon group may contain one or more heteroatoms, such as N, O, P, S, or halo; For attachment to a nucleic acid molecule, use.

[0142] 42. The use according to item 41, The 2',3'-O-ketal moiety (IV) has at least one of the characteristics described in any one of items 2 to 13; use.

[0143] 43. The use according to item 40 or 41, The nucleic acid molecule has at least one of the characteristics described in any one of items 14 to 22. use.

[0144] 44. The use according to any one of items 41 to 43, The addition of the 2',3'-O-ketal moiety (IV) to the nucleic acid molecule comprises at least one of the features described in any one of items 23 to 25. use.

[0145] 45. The use according to any one of items 41 to 44, the reactive functional moiety Y is a phosphoramidate group; use.

[0146] The present invention will be more specifically outlined by the following figures and examples. [Example]

[0147] [Example 1] 1. Materials and Methods 1.1 Synthesis of lipids and oligonucleotides The synthesis of lipids was performed according to standard methods. The synthesis of oligonucleotides and the conjugation of oligonucleotides to lipids was performed according to standard methods.

[0148] The following siRNA molecules against KRAS(G12C) were synthesized for the experiments described herein: Sense strand: 5' lipid - GUGGUAGUUGGAGCUUGUG- dtdt-3' Antisense strand: 5'-CACAAGCUCCAACUACCAC- dtdt -3'

[0149] The sense and antisense strands of the siRNA molecules consist of ribonucleotide building blocks (uppercase letters), except for two deoxyribonucleotide building blocks (lowercase letters) that form an overhang at the 3' end. The compound PRAMO-01 (see Figure 1) was used as the lipid.

[0150] The ribonucleotide building blocks in the double-stranded portions of the sense and antisense strands were modified with alternating 2′-O-methyl and 2′-fluoro ribonucleotide building blocks as follows: Sense strand: 5' Lipid - fG.mU.fG.mG.fU.mA.fG.mU.fU.mG.fG.mA.fG.mC.fU.mU.fG.mU.fG dtdt 3' Antisense strand: 5' mC.fA.mC.fA.mA.fG.mC.fU.mC.fC.mA.fA.mC.fU.mA.fC.mC.fA.mC dtdt 3' m = 2′-O-methyl, f = 2′-fluoro

[0151] 1.2 Intratumoral injection of PRAMO-01 conjugated siRNA into PDX peritoneal metastatic colon tumor NMRI nu / nu mice. Female PDX peritoneal metastatic colon tumor NMRI nu / nu mice (6-8 weeks old) were intratumorally injected with water for injection (WFI) (negative group) or 20 μg / tumor of PRAMO-01-conjugated siRNA dissolved in WFI. Three mice were typically injected per substance. However, one mouse in the negative group showed no tumor growth. The animals were sacrificed three days after injection (Figure 2).

[0152] Three days after injection, mice were sacrificed. Tumors were harvested, snap-frozen, and stored at -80°C. RNA extraction was performed in TRIzol RNA Isolation Reagent (ThermoFisher Scientific, Germany) using a Qiagen TissueLyser and RNeasy Kit (Qiagen). cDNA was transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit and random hexamer primers (MBI Ferments, St. Leon-Rot, Germany). For qPCR, the TaqMan KRAS assay, TaqMan actin B assay, and TaqMan Mastermix (ThermoFischer Scientific, Germany) were used according to the manufacturer's instructions. Relative expression of KRAS was normalized to actin B and ΔΔC t This was determined by the method (Figure 2).

[0153] [1.3 Intratumoral injection of PRAMO-01 conjugated siRNA into Lu7462, a NSCLC PDX model harboring heterozygous KRAS G12C in NMRI nu / nu mice] Female PDX NSCLC Lu7462 tumor-bearing NMRI nu / nu mice (6-8 weeks old) were intratumorally injected with either WFI (negative group) or PRAMO-01-conjugated siRNA dissolved in WFI at 60 μg / tumor (six mice per group). Animals were sacrificed 3 days after injection (Figure 3A and B).

[0154] The tumor volume and body weight of each animal were then measured at 0, 24, and 72 hours after injection.

[0155] [1.4 Statistical analysis] Data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). Silencing levels were normalized to the mean of the WFI control in each of at least two independent in vivo experiments. Data were analyzed using analysis of variance (ANOVA) for multiple comparisons.

[0156] [2. Results] 2.1 KRAS expression in a mouse model of colon cancer The results of KRAS gene expression in PDX peritoneal metastatic colon tumor NMRI nu / nu mice using negative control or PRAMO-01-conjugated siRNA are shown in Figure 2. Results were normalized to the negative control (100%). Mice were intratumorally injected with 20 μg / tumor of PRAMO-01-conjugated siRNA. Tissues were harvested 3 days after injection. Results are shown as the mean ± standard deviation of at least two independent experiments.

[0157] A significant inhibition of KRAS gene expression was observed, with residual expression in the control group being 4.5% compared to the negative group.

[0158] 2.2 Changes in body weight and tumor volume in lung cancer models The results of body weight measurements (g) at 0, 24, and 48 hours after intratumoral injection of water or 60 μg / tumor of PRAMO-01-conjugated siRNA in female PDX NSCLC Lu7462 tumor NMRI nu / nu mice are shown in Figure 3A.

[0159] No significant differences were observed in the body weight of control or siRNA-treated mice.

[0160] Tumor volume measurements (cm) at 0, 24, and 72 hours after intratumoral injection of water or 60 μg / tumor of PRAMO-01 conjugated siRNA in female PDX NSCLC Lu7462 tumor NMRI nu / nu mice. 3The results of the measurements are shown in Figure 3B.

[0161] A significant reduction in tumor growth was observed in siRNA-treated mice compared to controls.

[0162] Example 2: Therapeutic study testing the efficacy of in vivo intratumoral application of PRAMO-01-conjugated siRNA in the NSCLC PDX model Lu7462 harboring a G12C heterozygous KRAS mutation in NMRI nu / nu mice. 1. Materials and Methods 1.1 Synthesis of lipids and oligonucleotides Lipid synthesis was performed according to standard methods.

[0163] 1.2 Tumor model A lung cancer patient tumor xenograft model (NSCLC PDX model, Lu7462, heterozygous KRAS G12C mutation; grown subcutaneously in female NMRI nu / nu mice; Fichtner I. et al. Establishment of patient-derived non-small cell lung cancer xenografts as models for the identification of predictive biomarkers. Clin Cancer Res, 14: 6456-6468; 2008) was selected for this study.

[0164] JPEG2026503773000014.jpg45153

[0165] Six- to eight-week-old female NMRI nu / nu mice were maintained under pathogen-free conditions at 22 ± 2°C, 50 ± 10% relative humidity, and a 12-h light / dark cycle. Animals were housed in individually ventilated cages (IVCs; maximum of five mice / cage) located in an air-conditioned room. Mice had free access to food and acidified water. All animals were supplied by EPO GmbH.

[0166] [2. Dosage and Schedule of the Agent] When the tumors of the mice reached a volume of 0.04 - 0.1 cm 3 intratumoral injection of 20 μl of the reagent was initiated individually for each mouse (day 0, time point 0 hours). The injection into each tumor was administered by multiple injections (at least 4 injection sites per tumor) and was performed by EPO GmbH.

[0167] Reagent injection for each mouse / tumor was performed at four defined time points individually for each mouse: day 0 (0 hours), day 3 (72 hours), day 6 (144 hours), and day 9 (216 hours). This study ended 10 days after the first reagent injection.

[0168] [3. Start and End of the Treatment] The mice were assigned to specific groups and numbered from 1 to 32. The treatment was initiated on day 0 (0 hours) from the first reagent injection when the tumors of individual mice reached a volume of 0.04 - 0.1 cm 3 Only the tumors of 17 mice reached the defined volume, and the treatment was started separately for two test cohorts. However, one mouse in the control group treated with naked siRNA against KRAS (G12C) had to be sacrificed on day 8 of the test due to poor animal condition and ethical reasons (weight decreased from 31.4 g to 29.4 g, lethargy, hypothermia).

[0169] [4. Statistical Analysis] The data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, California, USA). In each of at least 4 independent in vivo experiments, the data were analyzed using one-way analysis of variance.

[0170] [5. Results] The growth inhibitory activity of the compounds was evaluated by determining tumor volume (TV). During the study period, tumor volume and body weight were measured daily from day 0 to day 10, and the mean tumor volume ratio (T / C) between the siRNA-treated group and the vehicle-treated group was calculated (see Figures 4-7).

[0171] Tumor volume and T / C were calculated according to the following formula: Tumor volume = (tumor width) × (tumor width) × (tumor length) ÷ 2 T / C = T÷C T: Mean estimated tumor volume of the compound-treated group C: Mean estimated tumor volume in the vehicle-treated group

[0172] During the experimental period, a significant decrease in tumor growth was observed in both lipid-conjugated and naked siRNA-treated mice, although a higher tumor reduction efficiency was observed with lipid-conjugated siRNA (see Figures 4-6).

[0173] Figure 8 shows Western blot analysis of tumors from PDX models (6-8 week old female NMRI nu / nu mice bearing patient-derived NSCLC tumor Lu7462).

Claims

1. 1. A nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, the nucleic acid molecule is directed against a target gene; used to inhibit expression of the target gene in a subject. Nucleic acid conjugates.

2. A nucleic acid conjugate according to claim 1 for the use according to claim 1, The nucleic acid conjugate has Formula (I) A, which includes at least one 2',3'-O-ketal moiety: where: X is a nucleic acid molecule directed against a target gene, B is a nucleobase, and R1 and R2 are each independently a hydrocarbon group; The hydrocarbon group may contain one or more heteroatoms, such as N, O, P, S, or halo; Here, R1 and R2 are specifically straight-chain or branched C ９ is an alkyl group, used to inhibit expression of the target gene in an organism, Nucleic acid conjugates.

3. A conjugate according to claim 1 or 2 for the use according to claim 1, the nucleobase B is a heterocyclic base, such as a purine or pyrimidine base, specifically selected from uracil, thymine, or analogs thereof; and / or the nucleobase B is a modified base, e.g., a base substituted with a hydrophobic moiety, a base substituted with a carbohydrate moiety, or a base substituted with a functional moiety; The hydrophobic moiety is specifically a terpene moiety, specifically C ５ -C ３０ The terpene portion, more specifically C ５ -C ２０ The terpene portion, more specifically C １５ a terpene moiety, or a sterol moiety, such as a cholesterol moiety; Conjugates.

4. A conjugate according to any one of claims 1 to 3 for the use according to claim 1, wherein B is a nucleobase of formula (IIa): wherein Z is CH or N and R3 is H; or wherein B is a nucleobase of formula (IIb): where Z is CH or N, and R3 is a terpene moiety, specifically C ５ -C ３０ The terpene portion, more specifically C ５ -C ２０ The terpene portion, more specifically C １５ Terpene moieties, for example, terpene moieties of formula (III): Conjugates.

5. A conjugate according to any one of claims 1 to 4 for the use according to claim 1, the nucleic acid molecule is an siRNA molecule; Conjugates.

6. A conjugate according to claim 5 for the use according to claim 1, The hydrophobic moiety, e.g., the 2',3'-O-ketal moiety, is added to the 5' end of the sense strand and / or the 5' end of the antisense strand of the siRNA molecule. Conjugates.

7. A conjugate according to any one of claims 5 to 6 for the use according to claim 1, The sense and / or antisense strand of the siRNA comprises a 3' overhang. Conjugates.

8. A conjugate according to claim 7 for the use according to claim 1, the 3' overhang has a length of 2 nucleotides and consists of a dT building block; Conjugates.

9. A conjugate according to any one of claims 5 to 8 for the use according to claim 1, The double-stranded portion of the siRNA molecule comprises modified ribonucleotide building blocks, such as 2'-O-methyl ribonucleotide building blocks and / or 2'-fluoro ribonucleotide building blocks. Conjugates.

10. A conjugate according to any one of claims 5 to 9 for the use according to claim 1, the double-stranded portion of the siRNA molecule is composed of modified ribonucleotide building blocks, such as 2'-O-methyl ribonucleotide building blocks and / or 2'-fluoro ribonucleotide building blocks; Conjugates.

11. A conjugate according to any one of claims 9 to 10 for the use according to claim 1, The 2'-O-methyl and 2'-fluoro ribonucleotide building blocks are arranged alternately on the sense and antisense strands. Conjugates.

12. A conjugate according to any one of claims 9 to 11 for the use according to claim 1, the double-stranded portion of the siRNA molecule is comprised of base pairs of a first nucleobase from a 2'-O-methylribonucleotide building block and a second nucleobase from a 2'-fluororibonucleotide building block; the first nucleobase is complementary to the second nucleobase; Conjugates.

13. A conjugate according to any one of claims 1 to 12 for the use according to claim 1, the nucleic acid molecule is directed against an oncogene; Conjugates.

14. A conjugate according to any one of claims 1 to 13 for the use according to claim 1, The nucleic acid molecule is directed against a RAS gene, such as a KRAS gene, a HRAS gene, or a NRAS gene; Conjugates.

15. A conjugate according to any one of claims 1 to 14 for the use according to claim 1, The nucleic acid molecule is directed against a wild-type or mutant RAS gene. Conjugates.

16. A conjugate according to any one of claims 1 to 15 for the use according to claim 1, the nucleic acid molecule is directed against a KRAS, HRAS, or NRAS gene mutated at positions 3, 12, 13, 14, 19, 33, 58, 59, 61, 117, 118, and / or 146; Conjugates.

17. A conjugate according to any one of claims 1 to 16 for the use according to claim 1, the nucleic acid molecule is directed against a mutant KRAS gene mutated at position 12 or 13, e.g., mutated G12C, G12D, G12V, G12R, G12A, G12S, G13D, and / or G13C; Conjugates.

18. A conjugate according to any one of claims 1 to 17 for the use according to claim 1, expression of the target gene is inhibited by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a control; Conjugates.

19. A conjugate according to any one of claims 1 to 17 for the use according to claim 1 or 18, for the treatment of colon cancer, in particular colon cancer associated with overexpression of a RAS gene, such as the KRAS gene, the HRAS gene, or the NRAS gene, more particularly colon cancer associated with mutations, and optionally also with overexpression, of the KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variants, Conjugates.

20. A conjugate according to any one of claims 1 to 17 for the use according to claims 1 or 18 to 19, for the treatment of lung cancer, e.g. non-small cell lung cancer (NSCLC), in particular lung cancer associated with overexpression of a RAS gene, e.g. KRAS gene, HRAS gene, or NRAS gene, more particularly lung cancer associated with mutations, and optionally also with overexpression, of KRAS G12C, G12D, G12V, G12R, G12A, and / or G13C variants, Conjugates.

21. A conjugate according to any one of claims 1 to 17 for the use according to any one of claims 1 or 18 to 20, The subject is a mammalian subject, particularly a human subject; Conjugates.

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