EP4658784A1

EP4658784A1 - Modified nucleic acid conjugates for inhibiting gene expression

Info

Publication number: EP4658784A1
Application number: EP24703557.9A
Authority: EP
Inventors: Ida SHAEF; Thomas Hiller; Merle Fuchs
Original assignee: Pramomolecular GmbH
Current assignee: Pramomolecular GmbH
Priority date: 2023-02-06
Filing date: 2024-02-06
Publication date: 2025-12-10
Also published as: WO2024165551A1; JP2026503773A

Abstract

The present invention refers to a nucleic acid conjugate comprising a hydrophobic moiety, e.g., a 2',3'-O ketal moiety for inhibiting the expression of a target gene.

Description

Modified nucleic acid conjugates for inhibiting gene expression

Description

The present invention refers to a nucleic acid conjugate comprising a hydrophobic moiety, e.g., a 2’,3’-O ketal moiety for inhibiting the expression of a target gene.

WO 2014/048969 describes nucleolipids and a process for the preparation of nucleolipids. Inter alia, nucleotides comprising a lipid 2’,3’-O ketal moiety are described. These nucleotides were found to have pharmaceutical activity in certain cancer cells. The delivery of nucleic acid molecules comprising 2’,3’-O-ketal moieties into target cells is, however, not disclosed.

WO 2022/144422 discloses nucleic acid conjugates comprising at least one 2’,3’-O- ketal moiety and their use for therapy and diagnostics. The content of this document is herein incorporated by reference in its entirety.

It is an object of the present invention to provide nucleic acid molecules, which are highly effective in reducing the target gene expression in a living organism.

The inventors have found that an siRNA molecule directed against the G12C variant of the KRAS gene molecule having attached thereto a lipid-modified 2’,3’-O ketal moiety was highly effective in inhibiting KRAS(G12C) expression or tumor growth in experimental patient derived xenograft (PDX) mouse models in vivo for colon tumor and lung tumor. Thus, therapeutic nucleic acid conjugates with improved characteristics are provided.

A first aspect of the present invention is a nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, which is directed against a target gene, for use in inhibiting the expression of the target gene in a subject.

In certain embodiments, the hydrophobic moiety comprises one, two or three hydrocarbon groups. In particular embodiments, the hydrophobic moiety comprises two hydrocarbon groups. In certain embodiments, a hydrocarbon group comprises at least 5 C-atoms, e.g., 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 certain embodiments, at least one hydrocarbon group is a linear hydrocarbon chain. In particular embodiments, all hydrocarbon groups are linear hydrocarbon chains.

In certain embodiments, the nucleic acid molecule of the conjugate is an siRNA molecule. In particular embodiments, the hydrophobic moiety is attached to the 5'-end of the sense strand of an siRNA molecule. In further particular embodiments, the hydro- phobic moiety is attached to the 5'-end of the antisense strand of an siRNA molecule.

In certain embodiments, the sense strand and/or the antisense strand of an siRNA comprises at least one 3'-overhang. In particular embodiments, the overhang has a length of 2 nucleotides and consists of dT building blocks.

In particular embodiments, the double-stranded portion of the siRNA molecule comprises modified ribonucleotide building blocks, e.g., 2'-O-methyl ribonucleotide building blocks and/or 2'-fluoro ribonucleotide building blocks. In further particular embodiments, the double-stranded portion of the siRNA molecule consists of modified ribonucleotide building blocks, e.g., 2'-O-methyl ribonucleotide building blocks and/or 2'- fluoro ribonucleotide building blocks.

In particular embodiments, the 2'-O-methyl and 2'-fluoro ribonucleotide building blocks are arranged in alternate succession on the sense strand and antisense strand. In further particular embodiments, the double-stranded portion of the siRNA molecule consists of base pairs of a first nucleobase from a 2'-O-methyl ribonucleotide building block and a second nucleobase from a 2 '-fluoro ribonucleotide building block wherein the first nucleobase is complementary to the second nucleobase, e.g., A-ll, ll-A, G-C, and C-G. In certain embodiments, the target gene is an oncogene. In particular embodiments, the target gene is a RAS gene, e.g., a KRAS gene, HRAS gene or NRAS gene.

The RAS genes encode RAS proteins that act as a cellular switch that is turned on by extracellular stimuli, resulting in the transient formation of the active, GTP-bound form of RAS, which activates different signalling pathways that regulate fundamental cell processes. Mutated RAS oncoproteins differ functionally from their normal counterparts in that the oncogenic forms prevent GAP from increasing the intrinsic catalytic rate of GTPase, thereby keeping RAS in its constitutively GTP-bound active state, which activates oncogenic pathways and cellular signal transduction.

In certain embodiments, the target gene is a wild-type RAS gene, e.g., a wild-type KRAS, HRAS, or NRAS gene. In these embodiments, the nucleic acid conjugate of the present invention may be used for the treatment of a disorder involving overexpression of the wild-type gene.

In certain embodiments, the target gene is a mutated RAS gene, e.g., a mutated KRAS, HRAS, or NRAS gene.

In particular embodiments, the target gene is a KRAS, HRAS, or NRAS gene mutated at position 3, 12, 13, 14, 19, 33, 58, 59, 61 , 117, 118 and/or 146, wherein the mutation particularly comprises an amino acid substitution. Exemplary mutations of the KRAS gene include but are not limited to G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61 H, Q61 L, Q61 R, A59E, A59G, A59T, K117N, K117R, K117E, A146T, A146P, and A146V. Exemplary mutations of the NRAS gene include but are not limited to G12D, G12C, G12S, G13R, G13V, K117R, Q61 H, Q61 L, Q61 K, Q61 R, A59D, A59T, and A146T.

In even more particular embodiments, the target gene is a mutated KRAS gene mutated at position 12 or 13, e.g., G12C, G12D, G12V, G12R, G12A, G12S, G13D, and/or In certain embodiments, the present invention encompasses administration of at least two different nucleic acid conjugate molecules, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 different nucleic acid conjugate molecules, directed against different target genes. In particular embodiments, the at least two different nucleic acid conjugate molecules are directed against different mutated forms of a target gene, e.g., different mutated forms of a RAS gene. In further particular embodiments, the at least two different nucleic acid conjugate molecules are directed against different mutated forms of the KRAS gene such as G12D, G12V, G12R and G12A.

In certain embodiments, the nucleic acid conjugate is used for the treatment of the following cancer types:

- Adrenal gland cancer, particularly adrenal cortical tumor

- Brain cancer, particularly glioblastoma multiforme or lower grade glioma,

- Head & neck cancer, particularly head and neck squamous cell carcinoma, papillary thyroid carcinoma, or salivary gland tumor;

- Endocrine cancer, particularly anaplastic thyroid carcinoma or follicular thyroid carcinoma;

- Thoracic cancer, particularly lung adenocarcinoma (e.g., NSCLC), lung squamous cell carcinoma, thymic carcinoma, or upper aerodigestive tract cancer

- Breast cancer, particularly invasive breast carcinoma;

- Core gastrointestinal cancer, particularly esophageal carcinoma, stomach adenocarcinoma, small intestine adenocarcinoma, colon adenocarcinoma, or rectal adenocarcinoma;

- Accessory gastrointestinal cancer, particularly liver hepatocellular carcinoma, gallbladder carcinoma, pancreatic adenocarcinoma, or biliary tract cancer;

- Genito-urinary cancer, particularly renal cell carcinoma, bladder urothelial carcinoma, or prostate adenocarcinoma;

- Gynaecological cancer, particularly ovarian cancer, uterine corpus endometrial carcinoma, cervical squamous carcinoma, endocervical adenocarcinoma, or endometrial cancer;

- Skin cancer, particularly skin cutaneous melanoma; - Haematopoietic/lymphoid cancer, particularly acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, or plasma cell myeloma;

- Bone cancer, particularly osteosarcoma, or malignant fibrous histiocytoma;

- Soft tissue cancer, particularly soft tissue sarcoma.

In certain embodiments, the nucleic acid conjugate is used for the treatment of colon cancer, e.g., colon adenocarcinoma. In particular embodiments, the colon cancer is associated with an overexpression of a RAS gene, e.g. , the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the colon cancer is associated with a mutation and optionally an overexpression the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the colon cancer is associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

In certain embodiments, the nucleic acid conjugate is used for the treatment of lung cancer e.g., lung adenocarcinoma, especially non-small cell lung cancer (NSCLC), or lung squamous cell carcinoma. In particular embodiments, the lung cancer is associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the lung cancer is associated with a mutation and optionally an overexpression the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the lung cancer is associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

In certain embodiments, the nucleic acid conjugate is used for the treatment of a brain cancer, particularly glioblastoma multiforme or lower grade glioma. In particular embodiments, the brain cancer is associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the brain cancer is associated with a mutation and optionally an overexpression the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the brain cancer is associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant. In certain embodiments, the nucleic acid conjugate is used for the treatment of a pancreas cancer, particularly pancreatic adenocarcinoma. In particular embodiments, the pancreas cancer is associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the pancreas cancer is associated with a mutation and optionally an overexpression the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the pancreas cancer is associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

In certain embodiments, the nucleic acid conjugate is used for the treatment of a gynaecological cancer, particularly ovarian cancer. In particular embodiments, the gynaecological cancer is associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the gynaecological cancer is associated with a mutation and optionally an overexpression the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the gynaecological cancer is associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

In certain embodiments, the nucleic acid conjugate is used for the treatment of a skin cancer, particularly skin cutaneous melanoma. In particular embodiments, the skin cancer is associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the skin cancer is associated with a mutation and optionally an overexpression the KRAS gene, HRAS gene or NRAS gene. In further particular embodiments, the skin cancer is associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

In certain embodiments, administration, e.g., single or multiple administration of the nucleic acid conjugate will inhibit 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. In certain embodiments, the subject is a mammalian subject, particularly a human subject.

In a further aspect of the present invention, the nucleic acid conjugate is a nucleic acid conjugate of the formula (I) comprising at least one 2’,3’-O-ketal moiety: wherein

X is a nucleic acid molecule which is directed against a target gene,

B is a nucleobase, and

R1 and R2 are independently from each other hydrocarbon groups wherein said hydrocarbon groups optionally comprises one or more heteroatom(s) such as N, 0, P, S, or halo.

A further aspect of the present invention relates to a method for inhibiting the expression of the target gene in a subject comprising administering to said subject a therapeutically effective amount of a nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, particularly of a nucleic acid conjugate of the formula (I) comprising at least one 2’,3’-O-ketal moiety: wherein

X is a nucleic acid molecule which is directed against said target gene,

B is a nucleobase, and

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

The nucleic acid conjugate of the formula (I) comprises a nucleic acid molecule X, to which at least one 2’,3’-O-ketal moiety is attached.

The term “nucleic acid molecule" relates to any type of oligonucleotide or polynucleotide comprising at least two nucleotide building blocks. The nucleic acid molecule may comprise ribonucleotide building blocks, 2’-desoxyribonucleotide building blocks, modified nucleotide building blocks or any combinations thereof. In certain embodiments, the nucleic acid molecule is an RNA molecule, optionally comprising at least one modified nucleotide building block and/or at least one 2’-desoxyribonucleotide building block, a DNA molecule, optionally comprising at least one modified nucleotide building block and/or at least one ribonucleotide building block, or a nucleic acid analogue molecule consisting of modified nucleotide building blocks.

As used herein, the term “nucleic acid analogue molecule’’, in particular DNA or RNA molecule analogue, refers to a molecule which is analogues, i.e. structurally similar to a naturally occurring RNA or DNA molecule. In general, naturally occurring nucleic acids are chains of nucleotides, which are composed of three elements: a backbone moiety, in particular a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these elements altered, such as modified nucleobases or locked nucleic acids (LNAs). Corresponding analogues are known to the person skilled in the art. An analogue may confer, among other things, different base pairing and base stacking properties.

The 2’,3’-O ketal moiety comprises a nucleobase B. The term “nucleobase" typically relates to a cyclic, e.g. mono- or bicyclic, saturated, unsaturated, aromatic or heteroaromatic base comprising at least one N atom wherein the nucleobase is capable of forming a base pair via hydrogen bonds with a complementary nucleobase, particularly with a naturally occurring nucleobase. The nucleobase may be any naturally occurring or non-naturally occurring nucleobase, e.g. any naturally occurring or non-naturally occurring purine or pyrimidine base such as adenine, cytosine, guanine, thymine, or uracil or a modified nucleobase, e.g. a modified adenine, cytosine, guanine, thymine, or uracil base.

The nucleobase of the 2’,3’-O ketal moiety may be a “modified nucleobase" . This term includes any type of modified nucleobase, e.g. a nucleobase substituted with a hydro- phobic moiety, a nucleobase substituted with a carbohydrate moiety and/or a nucleobase substituted with a functional moiety.

The hydrophobic moiety may be selected from moieties having at least 5 C-atoms, particularly at least 10 C-atoms.

In certain embodiments, the hydrophobic moiety is selected from substituted or unsubstituted acyclic or cyclic terpene moieties. For example, the hydrophobic moiety may be a C5-C30 terpene moiety, particularly a C5-C20 terpene moiety, and more particularly a C15 terpene moiety, which may be cyclic or acyclic, which may be saturated or unsaturated, and/or optionally be substituted or interrupted with heteroatom(s) or functional group(s), or a sterol moiety, e.g. a cholesterol moiety. Further, 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 a C5-C30 carboxylic acid moiety, e.g. 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.

Further, 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-C30 alcohol, ketone, or amide moiety, e.g. a tocopherol moiety, a tocopheryl succinate moiety, a retinol moiety, a retinal moiety, a spermine moiety, or a spermidine moiety.

In certain embodiments, the hydrophobic moiety is selected from and/or wherein

R and R’ are independently selected from C1-C30 alkyl, preferably C5 to C25-alkyl, n is an integer ranging 1 to 6, preferably n is 1 or 2 and a is an integer ranging from 1 to 20, preferably 2 to 18, more preferably 6 to 16. The carbohydrate moiety may be a mono-, oligo- or polysaccharide moiety, including modified carbohydrates, e.g. acetylated carbohydrate moieties. For example, the carbohydrate moiety may be a GalNAc moiety.

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

The conjugate of formula (I) comprises a 2’,3’-O-ketal moiety wherein the 2’-OH group and the 3’-OH group of the ribose moiety have been converted to a ketal group comprising substituents R1 and R2.

R1 and R2 are independently from each other hydrocarbon groups wherein said hydrocarbon groups optionally comprise one or more heteroatom(s) such as N, 0, P, S, or halo.

R1 and R2 may be saturated, mono- or polyunsaturated acyclic linear or branched hydrocarbon groups, or saturated, mono- or polyunsaturated, aromatic or heteroaromatic cyclic hydrocarbon groups, which may be interrupted by or substituted with one or more heteroatom(s).

In certain embodiments, R1 and/or R2 are independently from each other C5-21 hydrocarbon groups, particularly C5-11 hydrocarbon groups and more particularly C9 hydrocarbon groups wherein said hydrocarbon groups optionally comprise one or more heteroatoms such as N, 0, P, S, or halo, e.g. F, Cl, Br, or I.

In further embodiments, R1 and/or R2 are independently from each other linear or branched alkyl groups, linear or branched alkenyl groups, and linear or branched al- kynyl groups wherein said alkyl, alkenyl or alkynyl groups optionally comprise one or more heteroatoms such as N, 0, P, S, or halo. In still further embodiments, R1 and/or R2 are independently from each other cyclic moieties, i.e. moieties comprising at least one cyclic structure, e.g. a mono-, bi- or tricyclic structure. The cyclic moiety may be a carbocyclic or heterocyclic moiety, e.g. 5-18 ring atoms wherein at least one C-atom may be replaced by at least one heteroatom selected from N, 0, and S.

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

In still further embodiments, R1 and/or R2 comprise independently from each other, a marker group, e.g. a fluorescence, luminescence, or radioactive marker group.

In still further embodiments, R1 and/or R2 comprise independently from each other, a nucleic acid molecule as herein defined, e.g. a nucleic molecule having up to 50 and particularly from 5 to 25 nucleotide building blocks.

In particular embodiments, R1 and R2 are linear or branched C9 alkyl groups, more particularly linear C9 alkyl groups.

In a specific embodiment, the conjugate comprises a nucleobase B, which is an uracil base of the formula (Ila): wherein Z is CH or N and R3 is H.

In this embodiment, R1 and R2 are preferably linear C9 alkyl groups. In a further specific embodiment, the conjugate comprises a nucleobase B, which is an uracil base of the formula (lib): wherein Z is CH or N and R3 is a hydrophobic moiety, e.g. terpene moiety, particularly a C5-C30 terpene moiety, more particularly a C5-C20 terpene moiety, and even more particularly a C15 terpene moiety, e.g. a terpene moiety of the formula (III):

In this embodiment, R1 and R2 are preferably linear C9 alkyl groups.

In a still further specific embodiments, the 2', 3'-O-ketal moiety attached to the conjugate is derived from the compound PRAMO-01 or the compound PRAMO-02 as shown in Fig. 1 .

In certain embodiments, the nucleic acid molecule of the nucleic acid conjugate as described herein above, e.g. the nucleic acid molecule X, has a strand length of at least about 5 nucleotide building blocks and up to 1 ,000 or more nucleotide building blocks. In further embodiments, the nucleic acid molecule has a strand length between about 5 to about 100 nucleotide building blocks, between about 10 and about 50 nucleotide building blocks, between about 12 and about 40 nucleotide building blocks and particularly between about 15 and about 30 nucleotide building blocks.

The term "nucleic acid molecule" encompasses single-stranded, double-stranded, triple-stranded, and quadruple-stranded nucleic acid molecules, e.g. DNA molecules or RNA molecules and analogues thereof. Further, the nucleic acid molecule may comprise at least one modified nucleotide building block as described below.

In one embodiment, the nucleic acid molecule is a DNA molecule, which may comprise at least one modified nucleotide building block. The term "DNA molecule" encompasses single-stranded, and multi-stranded, e.g. double-stranded, triplestranded, or quadruple-stranded DNA molecules, particularly single- or double stranded DNA molecules. Multi-stranded, e.g. double-stranded DNA molecules may comprise strands having the same length or strands having different lengths. In multistranded, e.g. double-stranded DNA molecules, the individual strands may be present as separate molecules or covalently connected via a single-stranded loop or via heterologous linker.

The term "DNA molecule" encompasses molecules consisting of natural DNA building blocks, i.e. 2'-deoxyribonucleotide building blocks, and molecules comprising at least one 2'-deoxyribonucleotide building block and at least one modified nucleotide building block and/or at least one ribonucleotide building block.

In a further embodiment, the nucleic acid molecule is an RNA molecule, which may comprise at least one modified building block and/or at least one 2'- deoxyribonucleoside building block. The term “RNA molecule" encompasses singlestranded, or multi-stranded, e.g. double-stranded, triple-stranded, and quadruple- stranded RNA molecules, particularly single- or double stranded RNA molecules. Multistranded RNA molecules may comprise strands having the same length or strands having different lengths. For example, double-stranded RNA molecules may be blunt- ended or may have at least one overhang, e.g. at least one 3'-overhang. In multistranded, e.g. double-stranded RNA molecules, the individual strands may be present as separate molecules or covalently connected via a single-stranded loop or via a heterologous linker.

The term “RNA molecule" encompasses molecules consisting of natural RNA building blocks, i.e. ribonucleotide building blocks, and molecules comprising natural RNA building blocks and at least one modified nucleotide building block and/or at least one 2 '-deoxyribonucleoside building block.

The term “nucleotide building block’ relates to a moiety, which can be incorporated into a nucleic acid molecule as herein described and 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” may encompass any type of nucleobase as herein described above for the nucleobase B of formula (I). The sugar moiety may encompass a ribose moiety or a 2’-deoxyribose moiety including any modification thereof. The backbone moiety may encompass a phosphoester group forming an internucleosidic linkage between two nucleotide building blocks including any modification thereof. The nucleic acid molecule X may comprise modified nucleotide analogue blocks comprising a nucleobase modification, a sugar modification and/or a backbone modification.

In certain embodiments, the nucleic acid molecule comprises at least one modified nucleotide building block, particularly selected from:

(a) a nucleobase-modified building block;

(b) a sugar-modified building block;

(c) a backbone-modified building block and

(d) any combination thereof.

The term “nucleobase-modified building block” relates to a nucleotide building comprising a modified nucleobase. The term “modified nucleobase” may encompass any type of modified nucleobase as herein described above for the nucleobase B of formula (I).

The term “sugar-modified building block” “relates to a nucleotide building comprising a modified sugar. The term “sugar” typically relates to a ribose or 2’-deoxyribose moiety and includes any type of modified sugar moiety. In certain embodiments, a sugar-mod- ified nucleotide building block is selected from a nucleotide building block wherein the ring of the ribose moiety and/or the substituents on the ring are modified.

In certain embodiments, the modified nucleotide building block is a 2’-modified nucleotide building block wherein the 2'-OH substituent of ribose moiety is replaced by 2'- R4 wherein 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 al- kynyl, amino including mono- or disubstituted amino, e.g., C1-C5 alkyl, C2-C5 alkenyl or C2-C5 alkynyl substituted amino wherein each alkyl, alkenyl or alkynyl group is optionally substituted with OH, halo, cycloalkyl, cycloalkenyl, (hetero)aryl, e.g. phenyl, 0- alkyl, S-alkyl, and/or amino.

In certain embodiments, the modified nucleotide building block is a bridged, e.g. a 2’- 4'-bridged modified nucleotide building block, wherein the bridge typically has a length of 2-5 atoms, particularly 2-3 atoms including C-atoms and optionally heteroatoms such as 0, N or S. In a particular embodiment, the modified building block is a locked nucleic building block having a 2'-O-CH2-4' bridge.

In certain embodiments, the modified nucleotide building block is building block wherein the ring of the ribose moiety is modified, e.g. a morpholino building block, a thio-ribose building block, a 6-membered pyranose building block or a peptidic nucleic acid (PNA) building block.

The term “backbone-modified building block” relates to a nucleotide building comprising a modified internucleosidic linkage. In a backbone-modified nucleotide building block, the phosphoester group connecting adjacent building blocks is replaced by modified internucleosidic linkage, e.g. a phosphorothioate linkage, an alkyl phosphonate, e.g. methyl phosphonate linkage, and a borano phosphate linkage.

The nucleic acid molecule is preferably selected from nucleic acid molecules, which are suitable for pharmaceutical applications. Preferred nucleic acid molecules include mRNA molecules comprising coding sequences, particularly mRNA molecules comprising protein-coding sequences, RNA molecules capable of RNA interference such as 16-27-mer siRNAs, particularly 21 -mer siRNAs, blunt siRNAs, sisiRNAs, shRNAs, asiRNAs, aiRNAs, Fork siRNAs, 27-mer siRNA, Dumbbell siRNA, 16 mer- siRNAs, ss-siRNAs; microRNA molecules and antagomirs thereof, e.g. pre-miRNA mimics; RNA molecules capable of gene-editing, e.g. constituents of the CRISPR/Cas complex such as tracrRNAs, crRNAs, sgRNAs; antisense DNAs or RNAs; triplex or quadruplex forming nucleic acid molecules; CpG-oligonucleotides, TTAGGG- oligonucleotides, aptamers, ribozymes, or DNAzymes or precursors or modifications of such molecules.

In certain embodiments, the nucleic acid molecule is an RNA molecule optionally comprising at least one modified building block and/or at least one DNA building block, a DNA molecule optionally comprising at least one modified building block and/or at least one RNA building block, or a nucleic acid analogue molecule consisting of modified building blocks.

In further embodiments, the nucleic acid molecule is a double-stranded RNA molecule optionally comprising at least one modified building block and optionally having 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 particular embodiments, the RNA molecule is a siRNA molecule.

In further embodiments, the nucleic acid molecule is a single-stranded DNA molecule optionally comprising at least one modified building block and/or at least one ribose building block, particularly an antisense molecule.

The nucleic acid conjugate, e.g., the nucleic acid conjugate of the formula (I) may be prepared by attaching a hydrophobic moiety, e.g., a 2’-3’-O-ketal moiety (IV) to the nucleic acid molecule, wherein the 2’-3’-O-ketal moiety is of the formula (IV): wherein

Y is a reactive functional moiety, e.g. a phosphoamidate group or another group suitable in the chemical synthesis of nucleic acid molecules, in particular nucleic acid molecule backbones,

B is a nucleobase, and

R1 and R2 are independently from each other hydrocarbon groups wherein said hydrocarbon groups optionally comprise one or more heteroatoms such as N, 0, P, S, or halo.

In a still further specific embodiments, the 2', 3'-0-ketal moiety (IV) is the compound PRAMO-01 or the compound PRAMO-02 as shown in Fig. 1.

As used herein, the term “phosphoamidate” may also comprise “phosphoamidate variants” used and known by the skilled person for nucleic acid molecule synthesis.

The synthesis of the 2’,3’-O-ketal moiety (IV) is described in WO 2014/048969, the disclosure of which is herein incorporated by reference. The attachment of the 2’,3’-O- ketal moiety (IV) or any other hydrophobic moiety to the nucleic acid molecule may be performed during or after synthesis of the nucleic acid molecule, e.g. according to standard methods in solid phase nucleic acid synthesis.

In certain embodiments, a hydrophobic moiety, e.g. a 2’,3’-O-ketal building block is attached to the 5'-terminus and/or to the 3'-terminus of at least one strand of the nucleic acid molecule. In specific embodiments, a hydrophobic moiety, e.g. a 2’,3’-O-ketal building block is attached to the 5'-terminus of the sense strand of a double-stranded siRNA molecule or to the 5'-terminus of an antisense molecule.

In further embodiments, a hydrophobic moiety, e.g. a 2’,3’-O-ketal building block is attached to a nucleobase within a strand of the nucleic acid molecule.

In further embodiments, there is a plurality, e.g. 2 hydrophobic moieties, e.g. 2’,3’-O- ketal building blocks attached to the nucleic acid molecule.

A further aspect of the invention relates to the use of the nucleic acid conjugate described herein, e.g. the conjugate of the formula (I) in medicine, e.g., for use in human medicine or veterinary medicine, particularly for use in human medicine and in vivo animal research. In certain embodiments, the medical use comprises administering the conjugate to a subject in need thereof, particularly to a human subject. In further embodiments, the conjugate is administered to a target cell or a target organ ex vivo and the target cell or target organ is subsequently introduced into a subject in need thereof, particularly into a human subject. In still further embodiments, the conjugate is administered to an oocyte or embryo, wherein the use of human stem cells or human embryos for industrial commercial purposes is excluded. If the inventive conjugate is administered to an oocyte or embryo, it is preferred that the oocyte's or embryo's germline is not genetically modified, but only a disease is treated, such as a genetic disease. According to a preferred embodiment, the inventive conjugate is administered to a non-human oocyte or a non-human embryo.

The present invention provides an efficient method of mediating target -specific nucleic acid modifications in a cell, tissue, organ, or an organism comprising the steps:

(a) contacting a cell, tissue, organ, or organism with the conjugate of the invention, and

(b) mediating a target-specific nucleic acid modification effected by the nucleosidic component of the conjugate towards a target nucleic acid. 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 micro-organism, or a target cell, or a plurality of target cells within a multicellular organism , such as a target tissue and/or target organ. The target cell, target tissue and/or target organ is preferably a mammalian, including a human. The target organism is preferably a mammalian organism, e.g. 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.

The introducing into an organism may comprise any type of administration including systemic or local administration, e.g. by injection or infusion. Exemplary types of administration include aural, buccal, endobronchial, enteral, epidural, inhalative, instillation into the bladder, intra-arterial, intra-articular, intragastric, intragluteal, intracardiac, intracutaneous, intralumbar, intralymphatic, intramammary, intramuscular, intranasal, intraneural, intraocular, intraosseous, intraperitoneal, intrapleural, intrapulmonary, intraruminal, intrathecal, intratracheal, intraurethral, intrauterine, intravenous, intraventricular, intravitreal, oral, peroral, parenteral, peridural, perineural, percutaneous, rectal, retrobulbar, subconjunctival, subcutaneous, sublingual, topical, transdermal, transmucosal and/or vaginal administration.

Mediating step (b) preferably comprises a modification of a target nucleic acid, e.g. , by RNA interference when using a siRNA conjugate, or by inhibition of mRNA transcription when using an antisense molecule conjugate.

The present invention also provides a pharmaceutical composition comprising a conjugate as described above as the active agent together with a suitable carrier. For diagnostic or therapeutic applications, the pharmaceutical composition may be in the form of a solution, e.g. a solution for infusion or injection, a cream, ointment, tablet, suspension, powder, or the like. The composition may be administered in any suitable way, e.g. by parenteral administration, e.g. injection or infusion, by transmucosal application, or by transdermal application, or by oral, topical, nasal, rectal application, etc. Local applications are particularly preferred, such as in the abdominal cavity or in the peritoneum during surgery. Further preferred are applications in the vicinity of tumors or in the eye, in particular for siRNAs conjugates according to the invention.

The pharmaceutical composition may comprise the conjugate as an active agent or a 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 an encapsulated form or in non-encapsulated form, together 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.

In certain embodiments, the conjugate of the present invention may be used in the regulation, e.g., the downregulation or the upregulation of a gene of interest in a target cell, target tissue, target organ or a target organism. The gene of interest may be an endogenous gene or a gene from an exogenous pathogen, particularly a viral or bacterial gene or an endogenous disease-associated gene such as an oncogene or an autoimmune disease- or an allergic disease-associated gene.

In further embodiments, the conjugate of the present invention may be used in the introduction and optionally expression of coding, e.g. protein coding nucleic acid molecules such as mRNA molecules in a target cell or a target organism.

A specific aspect of the invention relates to the use of the conjugate for a target cell-, target-issue and/or target organ-specific delivery of a nucleic acid molecule, particularly of a therapeutic nucleic acid molecule as herein described. An especially preferred embodiment of the present invention relates to the use of a hydrophobic moiety, e.g. a 2’,3’-O-ketal building block as herein described being attached to the 5'-terminus of the sense strand of a double-stranded siRNA molecule or to the 5'-terminus of an antisense molecule for a target cell, target tissue and/or target organ specific delivery, in particular in vivo delivery. Thus, the use of siRNA conjugates for a target cell-, target- issue and/or target organ-specific delivery of a siRNA molecule, in particular a therapeutic siRNA molecule, is in particular preferred.

For example, the target cell may be a lung cell, a heart cell, a kidney cell, a liver cell, a pancreas cell, a colon cell, a muscle cell, a neural cell, a stomach cell, a small intestine cell, large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, a cell of the eye, a skin cells, or a brain cell. As used herein, “tissue” refers to an organic material consisting of a group of similarly or differently differentiated cells, such as target cells, that share a common function or structure.

Still a further aspect of the invention relates to an in vitro use of the conjugate for the delivery of a nucleic acid molecule into a target cell, target tissue or target organ. The target cell may be selected from any type of cell into which a nucleic acid molecule may be delivered, e.g. animal cells such as mammalian cells, bird cells or insect cells, plant cells, fungal cells, protozoan cells, bacterial cells and archaea cells. In particular embodiments, the target cell is a human cell.

A specific embodiment of this aspect relates to the use of the conjugate in the downregulation of a gene in a target cell, target tissue and/or target organ.

In particular, the invention provides a target cell-specific delivery, a target tissue specific delivery and/or a target organ specific delivery of a nucleic acid molecule in vitro. For example, the target cell may be a lung cell, a heart cell, a kidney cell, a liver cell, a pancreas cell, a colon cell, a muscle cell, a neural cell, a stomach cell, a small intestine cell, large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, a cell of the eye, a skin cells, or a brain cell.

A still further aspect of the present invention relates to the use of a 2’,3’-O-ketal moiety (IV) as herein described for the attachment to a nucleic acid molecule.

Further aspects of the invention are as described in the following items of the specification. Items A nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, which is directed against a target gene, for use in inhibiting the expression of the target gene in a subject. A nucleic acid conjugate of the formula (I) comprising at least one 2’,3’-O-ketal moiety: wherein

X is a nucleic acid molecule which is directed against a target gene,

B is a nucleobase, and

R1 and R2 are independently from each other hydrocarbon groups wherein said hydrocarbon groups optionally comprises one or more heteroatom(s) such as N, 0, P, S, or halo, for use in inhibiting the expression of the target gene in an organism. The conjugate of item 2, wherein R1 and R2 are independently from each other C5-21 hydrocarbon groups, particularly C5-11 hydrocarbon groups and more particularly C9 hydrocarbon groups wherein said hydrocarbon groups optionally comprise one or more heteroatoms such as N, 0, P, S, or halo. The conjugate of item 2 or 3, wherein R1 and R2 are independently from each other linear or branched alkyl groups, linear or branched alkenyl groups, and linear or branched alkynyl groups wherein said alkyl, alkenyl or alkynyl groups optionally comprise one or more heteroatoms such as N, 0, P, S, or halo. The conjugate of any one of items 2-4, wherein R1 and R2 are linear or branched C9 alkyl groups. The conjugate of any one of items 2-5, wherein the nucleobase B is a mono- or bicyclic aromatic or heteroaromatic base comprising at least one N atom. The conjugate of any one of items 2-6, wherein the nucleobase B is a heterocyclic base, e.g. a purine or pyrimidine base, particularly selected from uracil, thymine, or an analogue thereof. The conjugate of any one of items 2-7, wherein 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 conjugate of item 8, wherein the hydrophobic moiety is a terpene moiety, particularly a C5-C30 terpene moiety, more particularly a C5-C20 terpene moiety, and even more particularly a C15 terpene moiety, or a sterol moiety, e.g. a cholesterol moiety. The conjugate of item 8, wherein the carbohydrate moiety is a GalNAc group. The conjugate of item 8, wherein the functional moiety is a click-functional moiety such as an alkyne group or an azide group, or a such as an alkyne group or an azide group, or an ether, ester, amide, carboxylic acid, thioester, thioamide, or thioether group.

The conjugate of any one of items 2-7, wherein B is a nucleobase of the formula (Ila): wherein Z is CH or N and R3 is H.

The conjugate of any one of items 2-9, wherein B is a nucleobase of the formula (lib): wherein Z is CH or N and R3 is a terpene moiety, particularly a C5-C30 terpene moiety, more particularly a C5-C20 terpene moiety, and even more particularly a C15 terpene moiety, e.g. a terpene moiety of the formula (III): The conjugate of any one of the preceding items, wherein the nucleic acid molecule has a strand length of at least 5 nucleotide building blocks and up to 1 ,000 or more nucleotide building blocks, between 5 to 100 nucleotide building blocks, between W and 50 nucleotide building blocks, between 12 and 40 nucleotide building blocks and particularly between 15 and 30 nucleotide building blocks. The conjugate of any one of the preceding items, wherein the nucleic acid molecule comprises at least one modified nucleotide building block, which is particularly selected from:

(a) a base-modified nucleotide building block;

(b) a sugar-modified nucleotide building block;

(c) a backbone-modified nucleotide building block and

(d) any combination thereof. The conjugate of item 15, wherein the base-modified nucleotide building block is selected from a building block comprising a base substituted with a hydrophobic moiety, a nucleotide building block comprising a base substituted with a carbohydrate moiety and a nucleotide building block comprising a base substituted with a functional moiety. The conjugate of item 15, wherein the sugar-modified nucleotide building block is selected from a 2’-mod- ified ribose building block, a 2’-4'-bridged modified ribose building block, e.g., a locked nucleotide (LNA) building block, a morpholino building block and a pep- tidic nucleic acid (PNA) building block. The conjugate of item 15, wherein the backbone-modified nucleotide building block comprises a modified internucleosidic linkage, e.g., a phosphorothioate linkage, an alkyl phospho- nate, e.g., methyl phosphonate linkage, and a borano phosphate linkage. The conjugate of any one of the preceding items, wherein the nucleic acid molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. The conjugate of any one of items 1 -19, wherein the nucleic acid molecule is (i) an RNA molecule optionally comprising at least one modified nucleotide building block and/or at least one DNA building block, or (ii) a DNA molecule optionally comprising at least one modified nucleotide building block and/or at least one RNA building block, or (iii) a nucleic acid analogue molecule. The conjugate of item 20, wherein the nucleic acid molecule is a single-stranded, a double-stranded, a triple-stranded, or a quadruple-stranded RNA molecule, particularly a single or double stranded RNA molecule, wherein the RNA molecule optionally comprises at least one deoxyribonucleotide building block, at least one modified building block and optionally having at least one 3' -overhang, particularly a siRNA molecule. The conjugate of item 21 , wherein the nucleic acid molecule is a single-stranded, a double-stranded, a triple-stranded, or a quadruple-stranded RNA molecule, particularly a single or double stranded DNA molecule, wherein the DNA molecule optionally comprises at least one ribonucleotide building block, at least one modified building block, particularly an antisense molecule. he conjugate of any one of the preceding items, wherein the hydrophobic moiety, e.g., the 2’,3’-O-ketal building block is attached to the 5'-terminus and/or to the 3'-terminus of a strand of the nucleic acid molecule. he conjugate of item 23, wherein the hydrophobic moiety, e.g., the 2’,3’-O-ketal building block is attached to the 5'-terminus of the sense strand of a double-stranded siRNA molecule or to the 5'-terminus of an antisense molecule. he conjugate of any one of the preceding items, wherein the hydrophobic moiety, e.g., the 2’,3’-O-ketal building block is attached to a base within a strand of the nucleic acid molecule. The conjugate of any one of the preceding items for use in medicine, e.g. for use in human medicine or veterinary medicine, particularly for use in human medicine. The conjugate of any one of items 1 -25 for use according to item 26, wherein the conjugate is administered to a subject in need thereof, particularly to a human subject. The conjugate of any one of items 1 -25 for use according to item 26, wherein the conjugate is administered to a target cell or a target organ ex vivo and wherein the target cell or target organ is subsequently introduced into a subject in need thereof, particularly into a human subject. The conjugate of any one of items 1 -25 for use according to item 26, wherein the conjugate is administered to an oocyte or embryo, wherein the use of human stem cells or human embryos for industrial commercial purposes is excluded. The conjugate of any one of items 1 -25 for use according to any one of items 26-29, wherein the administration comprises a target cell- and/or target organspecific delivery. The conjugate of any one of items 1 -25 for use according to any one of items 26-30 in the upregulation or downregulation of a gene in a target cell or a target organism, e.g., an endogenous gene or a gene from an exogenous pathogen, particularly a viral or bacterial gene or an endogenous disease-associated gene such as an oncogene or an autoimmune disease- or allergic disease-associated gene. The conjugate of any one of items 1 -25 for use according to any one of items 26- 30 in the introduction and optionally expression of a coding nucleic acid molecule gene in a target cell or a target organism. The conjugate of any one of items 1 -25 for use according to any one of items 26-32 wherein the target cell is a lung cell, a heart cell, a kidney cell, a liver cell, a pancreas cell, a colon cell, a muscle cell, a neural cell, a stomach cell, a small intestine cell, large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, a cell of the eye, a skin cells, or a brain cell. In vitro use of the conjugate of any one of items 1 -25 for the delivery of a nucleic acid molecule into a target cell. The use of item 34, wherein the target cell is selected from animal cells such as mammalian cells, bird cells or insect cells, plant cells, fungal cells, protozoan cells, bacterial cells, and archaea cells. The use of item 34 or 35, wherein the target cell is a human cell. The use of any one of items 34-36 for the upregulation or downregulation of a gene in the target cell. The use of any one of items 34-36 for the introduction and optionally expression of a coding nucleic acid molecule gene in a target cell or a target organism. The use of any one of items 34-38 comprising a target cell-specific delivery. The use of any one of items 34-39, wherein the target cell is a lung cell, a heart cell, a kidney cell, a liver cell, a pancreas cell, a colon cell, a muscle cell, a neural cell, a stomach cell, a small intestine cell, large intestine cell, a rectum cell, a bladder cell, a bone cell, an adrenal gland cell, a cell of the eye, a skin cells, or a brain cell. Use of a 2’,3’-O-ketal moiety (IV): wherein

Y is a reactive functional moiety,

B is a nucleobase, and

R1 and R2 are independently from each other hydrocarbon groups wherein said hydrocarbon groups optionally comprise one or more heteroatoms such as N, 0, P, S or halo, for the attachment to a nucleic acid molecule. The use of item 41 , wherein the 2’,3’-O-ketal moiety (IV) comprises at least one feature of any one of items 2-13. The use of item 40 or 41 , wherein the nucleic acid molecule comprises at least one feature of any one of items 14-22. 44. The use of any one of items 41 -43, wherein the attachment of the 2’,3’-O-ketal moiety (IV) to the nucleic acid molecule comprises at least one feature of any one of items 23-25.

45. The use of any one of items 41 -44, wherein the reactive functional moiety Y is a phosphoamidate group.

The present invention shall be outlined in more detail by the following Figures and Examples.

Figure Legends:

Figure 1 : Structure and molar mass of 2',3'-O-ketal moieties PRAMO-01 and PRAMO-02 as phosphoamidites.

Figure 2: Inhibition of KRAS gene expression after administration of a lipid-modified anti-KRAS (G12C) siRNA molecule in PDX peritoneal metastasis colon-tumor NMRI nu/nu mice.

Figure 3: Changes in body weight (A) and tumor volume (B) after administration of a lipid-modified anti-KRAS (G12C) siRNA molecule in NSCLC PDX Lu7462 with heterozygote KRAS G12C NMRI nu/nu mice.

Figure 4: Analysis of tumor growth over time for all groups (day 0 - 10 of treatment). Measurement of tumor volume of PDX model (6-8-week-old female, NMRI nu/nu mice bearing patient derived NSCLC tumor-Lu7462, 0.04-0.1 cm³ at day 0). The animals were treated intratumorally with injection water (small square), self-delivering scrambled siRNA (downwards pointing black triangle), naked siRNA against mutation KRAS G12C (big square), or PRAMO-self-delivering siRNA against KRAS(G12C) (black dot). Animals were treated at day 0, 3, 6, and 9. At day 10, the animals were sacrificed. Tumors were measured mechanically every day (4 mice per group, one mouse in control group treated with naked siRNA against KRAS(G12C) had to be sacrificed at day 8 of the study due to the bad condition and ethical reasons, the data related to this mouse is therefore not included in this graph). Results are shown as 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: Analysis of tumor growth over time for vehicle vs treated group (day 0 - 10 of treatment). Measurement of tumor volume of PDX model (6-8-week-old female, NMRI nu/nu mice bearing patient derived NSCLC tumor-Lu7462, 0.04-0.1 cm³ at day 0). The animals were treated intratumorally with injection water (square) or PRAMO- self-delivering siRNA against KRAS(G12C) (black dot). Animals were treated at day 0, 3, 6, and 9. At day 10, the animals were sacrificed. Tumors were measured mechanically every day (4 mice per group). Results are shown as mean ± standard deviation of four independent experiments. Data were analyzed using one-way ANOVA test. * p < 0.05; ** p < 0.01.

Figure 6: Analysis of tumor volumes on day 0 vs day 10 of treatment. Measurement of tumor volume of PDX model (6-8-week-old female, NMRI nu/nu mice bearing patient derived NSCLC tumor-Lu7462, 0.04-0.1 cm³ at day 0). The animals were treated intratumorally with injection water (black dot), self-delivering scrambled siRNA (downwards pointing black triangle), naked siRNA against mutation KRAS G12C (upwards pointing black triangle), or PRAMO-self-delivering siRNA against KRAS(G12C) (black square). Animals were treated at day 0, 3, 6, and 9. At day 10, the animals were sacrificed. Tumors were measured mechanically every day (at least 4 mice per group, one of the mice in the control group treated with naked siRNA against KRAS(G12C) had to be sacrificed at day 8 of the study due to the bad condition of the animal and ethical reasons). Results are shown as mean ± standard deviation of four independent experiments. Data were analyzed using one-way ANOVA test. * p < 0.05; ** p < 0.01 .

Figure 7: Picture of explanted tumors at day 10 of treatment. Comparison of explanted tumors of PDX model (6-8-week-old female, NMRI nu/nu mice bearing patient derived NSCLC tumor-Lu7462, 0.04-0.1 cm³ at day 0). The animals were treated intratu morally with injection water or PRAMO-self-delivering siRNA against KRAS(G12C), self-delivering scrambled siRNA, or naked siRNA against KRAS (G12C). Animals were treated at day 0, 3, 6, and 9. At day 10, the animals were sacrificed, and the tumors were explanted.

Figure 8: Western blot analysis of isolated tumors at day 10 of treatment. Western blot analysis of tumors of PDX model (6-8-week-old female, NMRI nu/nu mice bearing patient derived NSCLC tumor-Lu7462). The animals were treated intratumorally with injection water or PRAMO-self-delivering siRNA against KRAS(G12C), self-delivering scrambled siRNA, or naked siRNA against KRAS (G12C). Animals were treated at day 0, 3, 6, and 9. At day 10, the animals were sacrificed, and the tumors were explanted (n=4).

Examples:

Example 1

1. Material and Methods

1.1 Synthesis of lipids and oligonucleotides

The synthesis of lipids were performed according to standard methods. Synthesis of oligonucleotides and conjugation of oligonucleotides to lipids were performed according to standard methods.

The following siRNA molecule against KRAS (G12C) was synthesized for the experiments described herein:

Sense strand: 5' Lipid- G-U-G-G-U-A-G-U-U-G-G-A-G-C-U-U-G-U-G- dtdt-3'

Antisense strand: 5'-C-A-C-A-A-G-C-U-C-C-A-A-C-U-A-C-C-A-C- dtdt -3'

The sense and antisense strands of siRNA molecule consist of ribonucleotide building blocks (uppercase letters) except 2 deoxyribonucleotide building blocks forming the overhangs at the 3'-ends (lowercase letters). As lipid, the compound PRAMO-01 (c.f. Fig. 1 ) was used.

The ribonucleotide building blocks in the double-stranded portions of the sense and antisense strands were alternately modified with 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.rnC.fA.mA.fG.mC.fU.mC.fC.mA.fA.mC.fll.mA.fC.mC.fA.mC dtdt 3' m = 2'-0-methyl, f = 2 '-fluoro

1 .2 Intratumoral injection of PRAMO-01 -conjuqated-siRNA into PDX peritoneal metastasis colon-tumor NMRI nu/nu mice

Female PDX peritoneal metastasis colon-tumor NMRI nu/nu mice (6-8-weeks-old) were injected intratumorally with water for injection (WFI) (negative group) or 20 pg/tu- mor PRAMO-01 -conjugated siRNA dissolved in WFI. In general, 3 mice per substance were prepared. However, a tumor growth in one mouse of the negative group could not be obtained. The animals were sacrificed at day 3 post-injection (Figure 2).

At day 3 post-injection, the mice were sacrificed. Tumors were collected, snap-frozen and stored at -80°C. RNA extraction was performed using Qiagen TissueLyser and RNeasy Kit (Qiagen) in TRIzol RNA Isolation Reagent (ThermoFisher Scientific, Germany). Using RevertAid H Minus First Strand cDNA Synthesis Kit and random Hexamer primers (MBI Ferments, St. Leon-Rot, Germany), cDNAs were transcribed. For qPCR, TaqMan KRAS Assay, TaqMan ACTINB Assay, and TaqMan Mastermix (ThermoFischer Scientific, Germany) were used according to the manufacturer’s instructions. The relative KRAS expression was normalized to Actin B and determined by AACt method (Figure 2).

1 .3 Intratumoral injection of PRAMO-01 -coniuqated-siRNAs into NSCLC PDX model Lu7462 with heterozyqote KRAS G12C in NMRI nu/nu mice

Female PDX NSCLC Lu7462-tumor NMRI nu/nu mice( 6-8-weeks-old) were injected intratumorally with WFI (negative group) or 60 pg/tumor PRAMO-01 -conjugated-siRNA dissolved in WFI (6 mice per group). The animals were sacrificed at day 3 post-injection (Fig. 3A and B). The tumor volume and body weight of each animal were then measured at 0 h, 24 h, and 72 h post injection.

1 .4 Statistical analysis

Data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). In each of the at least two independent in vivo experiments, the level of silencing was normalized to the mean of the WFI controls. Data were analyzed using ANOVA for multiple comparisons.

2. Results

2.1 KRAS expression in a colon cancer mouse model

The results of the expression of the KRAS gene in PDX peritoneal metastasis colontumor NMRI nu/nu mice using negative control or PRAMO-01 conjugated siRNA are shown in Figure 2. Results are normalized to the negative control (100%). Mice were injected intratumorally with 20 pg/tumor PRAMO-01 -conjugated siRNAs. Tissues were collected at day 3 post-injection. Results are shown as mean ± standard deviation of at least two independent experiments.

A substantial inhibition of KRAS gene expression was observed. The residual expression in the control group 4.5% versus the negative group.

2.2 Changes in body weight and tumor volume in a lung cancer model

The results of body weight measurements (g) in female PDX NSCLC Lu7462-tumor NMRI nu/nu mice at 0 h, 24 h, and 48 h after intratumoral injection of water or 60 pg/tumor of PRAMO-01 -conjugated-siRNA are shown in Figure 3A.

No significant differences in the body weight of control or siRNA treated mice were observed. The results of measurement of tumor volume measurements (cm³) in female PDX NSCLC Lu7462-tumor NMRI nu/nu mice at 0 h, 24 h, and 72 h after intratumoral injection of water or 60 pg/tumor of PRAMO-01 -conjugated-siRNA are shown in Figure 3B.

A substantial reduction in the tumor growth was observed in siRNA treated mice versus control.

Example 2: Therapeutic study to test the efficacy of in vivo intra-tumoral application of PRAMO-01 conjugated siRNA in NSCLC PDX model Lu7462, with G12C heterozygous KRAS mutation in NMRI nu/nu mice

1. Material and methods

1.1 Synthesis of Lipids and Oligonucleotides

The synthesis of lipid was performed according to standard methods.

1.2 Tumor Model

For this study, the patient-derived lung xenograft model (NSCLC PDX model, Lu7462, heterozygous KRAS G12C mutation; grown s.c. 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 chosen.

Table 1 : Selected PDX lung tumor model Lu7462.

Female 6 - 8-week-old NMRI nu/nu mice were kept under germ-free conditions, 22+/- 2°C, 50+/-10% relative humidity and 12 hour light-dark-cycle. The animals were housed in individually ventilated cages (IVC; max. 5 mice/cage) set in air-conditioned rooms. The mice had free access to food and acidified water. All animals were provided by EPO GmbH.

2. Drug dosage and schedule

When the tumor of a mouse reached a volume of 0.04 to 0.1 cm³, the intra-tumoral injection of 20 pl reagent started (day 0, time point Oh) individually for each mouse. The injection of each tumor was administered with multiple injections (at least 4 injection sites per tumor), performed by the EPO GmbH.

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

3. Start and end of treatment

Mice were assigned to the specific groups and numbered from 1-32. The treatments began with the first reagent injection at day 0 (0 h), when a tumor, for the individual mouse had reached a volume of 0.04 to 0.1 cm³. Only tumors of 17 mice reached the defined volume and the treatments started separately for two study cohorts. However, one of the mice in a control group treated with naked siRNA against KRAS(G12C) had to be sacrificed at day 8 of the study due to the bad condition of the animal and ethical reasons (body weight loss from 31 .4 g to 29.4 g, apathetic, drop in body temperature). 4. Statistical analysis

Data were analyzed using GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA, USA). In each of the at least four independent in vivo experiments, data were analyzed using one-way ANOVA.

5. Results

Growth inhibitory activity of the compounds was evaluated by determination of tumor volumes (TV). During the study, tumor volumes and body weights were measured daily, at days 0 to 10, and the ratio of the mean tumor volumes between the siRNA- treated group and the vehicle-treated group (T/C) was calculated (cf. Figures 4-7).

The tumor volume and T/C were calculated according to the following equations:

Tumor volume = (tumor width) x (tumor width) x (tumor length) - 2

T/C = T - C

T: Mean estimated tumor volume of the compound-treated group

C: Mean estimated tumor volume of the vehicle-treated group

A substantial reduction in the tumor growth during the experiments was observed for lipid-coupled- and naked siRNA-treated mice. However, a higher tumor reduction efficiency was observed for lipid-coupled siRNA (cf. Figures 4-6).

Figure 8 shows the Western Blot analysis of tumors of the PDX model (6-8-week-old female, NMRI nu/nu mice bearing patient derived NSCLC tumor-Lu7462).

Claims

1. A nucleic acid conjugate comprising a hydrophobic moiety conjugated to a nucleic acid molecule, which is directed against a target gene, for use in inhibiting the expression of the target gene in a subject.

2. The nucleic acid conjugate of claim 1 for the use of claim 1 , which has A the formula (I) comprising at least one 2’,3’-O-ketal moiety: wherein

X is a nucleic acid molecule which is directed against a target gene, B is a nucleobase, and

R1 and R2 are independently from each other hydrocarbon groups wherein said hydrocarbon groups optionally comprises one or more heteroatom(s) such as N, 0, P, S, or halo, wherein R1 and R2 particularly are linear or branched C9 alkyl groups, for use in inhibiting the expression of the target gene in an organism.

3. The conjugate of claim 1 or 2 for the use of claim 1 , wherein the nucleobase B is a heterocyclic base, e.g., a purine or pyrimidine base, particularly selected from uracil, thymine, or an analogue thereof, and/or wherein 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, wherein the hydrophobic moiety is particularly a terpene moiety, particularly a C5-C30 terpene moiety, more particularly a C5-C20 terpene moiety, and even more particularly a C15 terpene moiety, or a sterol moiety, e.g., a cholesterol moiety.

4. The conjugate of any one of claims 1 -3 for the use of claim 1 , wherein B is a nucleobase of the formula (Ila): wherein Z is CH or N and R3 is H; or wherein B is a nucleobase of the formula (lib): wherein Z is CH or N and R3 is a terpene moiety, particularly a C5-C30 terpene moiety, more particularly a C5-C20 terpene moiety, and even more particularly a C15 terpene moiety, e.g., a terpene moiety of the formula (III):

5. The conjugate of any one of claims 1 -4 for the use of claim 1 , wherein the nucleic acid molecule is an siRNA molecule.

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

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

8. The conjugate of claim 7 for the use of claim 1 , wherein the 3'-overhang has a length of 2 nucleotides and consists of dT building blocks.

9. The conjugate of any one of claims 5-8 for the use of claim 1 , wherein the double-stranded portion of the siRNA molecule comprises modified ribonucleotide building blocks, e.g., 2'-O-methyl ribonucleotide building blocks and/or 2 '-fluoro ribonucleotide building blocks.

10. The conjugate of any one of claims 5-9 for the use of claim 1 , wherein the double-stranded portion of the siRNA molecule consists of modified ribonucleotide building blocks, e.g., 2'-O-methyl ribonucleotide building blocks and/or 2 '-fluoro ribonucleotide building blocks.

11 . The conjugate of any one of claims 9-10 for the use of claim 1 , wherein the 2'- O-methyl and 2 '-fluoro ribonucleotide building blocks are arranged in alternate succession on the sense strand and antisense strand.

12. The conjugate of any one of claims 9-11 for the use of claim 1 , wherein the double-stranded portion of the siRNA molecule consists of base pairs of a first nucleobase from a 2'-O-methyl ribonucleotide building block and a second nu- cleobase from a 2'-fluoro ribonucleotide building block wherein the first nucleobase is complementary to the second nucleobase.

13. The conjugate of any one of claims 1 -12 for the use of claim 1 , wherein the nucleic acid molecule is directed against an oncogene.

14. The conjugate of any one of claims 1 -13 for the use of claim 1 , wherein the nucleic acid molecule is directed against a RAS gene, e.g., a KRAS gene, HRAS gene or NRAS gene.

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

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

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

18. The conjugate of any one of claims 1 -17 for the use of claim 1 , wherein the 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.

19. The conjugate of any one of claims 1 -17 for the use of claim 1 or 18 for the treatment of colon cancer, particularly colon cancer associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene, more particularly colon cancer associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

20. The conjugate of any one of claims 1 -17 for the use of claim 1 or 18-19 for the treatment of lung cancer e.g., non-small cell lung cancer (NSCLC), particularly lung cancer associated with an overexpression of a RAS gene, e.g., the KRAS gene, HRAS gene or NRAS gene, more particularly lung cancer associated with a mutation and optionally an overexpression the KRAS G12C, G12D, G12V, G12R, G12A and/or G13C variant.

21. The conjugate of any one of claims 1 -17 for the use of any one of claims 1 or 18-20, wherein the subject is a mammalian subject, particularly a human subject.