Dual Silence

Abstract

This disclosure relates to isolated nucleic acid molecules comprising at least two double-strand inhibitory ribonucleic acid (RNA) molecules adapted to be silenced by RNA interference, thereby silencing either the same gene or different genes and thereby regulating gene expression.

Description

Technical Field

[0001] The present disclosure relates to an isolated nucleic acid molecule comprising at least two double-stranded inhibitory ribonucleic acid (RNA) molecules adapted to be silenced by RNA interference of either the same gene or different genes, enhance the silencing, and thereby regulate gene expression.

Background Art

[0002] Techniques for specifically excising gene function are by introduction of double-stranded inhibitory RNA, also called small inhibitory or interfering RNA (siRNA), into cells, which results in the destruction of mRNA complementary to the sequence contained in the siRNA molecule. The siRNA molecule comprises two complementary strands of RNA (sense strand and antisense strand) that anneal to each other to form a double-stranded RNA molecule. The siRNA molecule typically, but not limited to, is derived from an exon of the gene to be removed. Many organisms respond to the presence of double-stranded RNA by activating a cascade leading to the formation of siRNA. The presence of double-stranded RNA activates a protein complex containing RNase III that processes the double-stranded RNA into smaller fragments (siRNA, approximately 21-29 nucleotides in length) that become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave the mRNA complementary to the antisense strand of the siRNA, thereby resulting in the destruction of the mRNA.

[0003] Typically, RNA silencing utilizes a single siRNA species directed at a single gene to silence its expression. It is known that siRNAs can be ligated to enable silencing of two or more genes. For example, WO2016205410 discloses oligonucleotides ligated together directly, via functional terminal substitution, or indirectly via a linker. Oligonucleotides can be directly bound to a linker. Such binding can be achieved, for example, through the use of a 3'-thionucleoside. WO2018145086 discloses oligonucleotides in the form of multimeric oligonucleotides having monomeric subunits of oligonucleotides linked by a covalent linker to reduce clearance by glomerular filtration. WO2013040429 discloses polyoligomeric complexes containing two or more targeted oligonucleotides ligated together by a cleavable linker. WO2015113922 discloses oligonucleotide conjugates in which two or more antisense oligonucleotides are covalently linked by a physiologically unstable linker, and bio-cleavable functional groups such as conjugate groups. WO2010141511 discloses nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, wherein the target-specific regions are complementary to a single target gene at two or more distinct nucleotide sites, and / or the target regions are complementary to two or more target genes or target sequences. WO2017188707 discloses a dicer substrate RNA nanostructure that demonstrates enhanced gene silencing effects. The RNA nanostructure contains multiple identical or different RNAi sequences within a single RNA nanostructure. The use of oligomeric nanostructures containing two or more siRNAs or antisense molecules is known in the art.

[0004] To enhance therapeutic effects, it is desirable to provide an alternative and simpler approach for delivering two or more inhibitory RNAs to cells for the purpose of silencing the expression of one or more gene targets.

[0005] Cardiovascular disease associated with hypercholesterolemia is a common condition with high rates of heart disease, mortality, and morbidity, and can be a result of diet, obesity, or genetically impaired genes. For example, mutations in the low-density lipoprotein receptor (LDL receptor) or apolipoprotein B (ApoB) in familial hypercholesterolemia. Cholesterol is essential for membrane biosynthesis in animal cells. Its lack of water solubility means that cholesterol is transported around the body in association with lipoproteins. Apolipoproteins are formed together with phospholipids, cholesterol, and lipids, and they facilitate the transport of lipids, such as cholesterol, through the bloodstream to different parts of the body. Lipoproteins are classified according to their size and can form HDL (high-density lipoprotein), LDL (low-density lipoprotein), IDL (intermediate-density lipoprotein), VLDL (very low-density lipoprotein), and LDL (very low-density lipoprotein) lipoproteins.

[0006] Familial hypercholesterolemia is an orphan disease resulting from elevated levels of LDL cholesterol (LDL-C) in the blood. This disorder is an autosomal dominant disorder that presents with both heterozygous (350–550 mg / dL LDL-C) and homozygous (650–1000 mg / dL LDL-C) states, leading to elevated LDL-C. Heterozygous familial hypercholesterolemia accounts for approximately 1:500 of the population. Homozygous states are far rarer, approximately 1:1,000,000. Normal LDL-C levels are in the 130 mg / dL range.

[0007] Hypercholesterolemia is particularly acute in pediatric patients, and if left undiagnosed early, can lead to accelerated coronary heart disease and premature death. If diagnosed and treated early, children can have a normal life expectancy. In adults, high LDL-C, due to either mutation or other factors, is directly associated with an increased risk of atherosclerosis, which can lead to coronary artery disease, stroke, or kidney problems. Lowering LDL-C levels is known to reduce the risk of atherosclerosis and related conditions. LDL-C levels can first be lowered by administering statins, which block the de novo synthesis of cholesterol by inhibiting HMG-CoA reductase. Some subjects may benefit from combination therapy, combining statins with other therapeutic agents such as ezetimibe, colestipol, or nicotinic acid. However, HMG-CoA reductase expression and synthesis adapt to statin inhibition and increase over time, and therefore, the beneficial effect is only temporary or limited after statin resistance has been established.

[0008] This disclosure relates to isolated nucleic acid molecules comprising at least two double-strand inhibitory ribonucleic acid (RNA) molecules adapted to be silenced by RNA interference of either the same gene or different genes, to enhance the silencing, and thereby to regulate gene expression. [Overview of the Initiative]

[0009] According to one aspect of the present invention, i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand designed with reference to a nucleotide sequence containing the gene to be silenced, and an antisense strand, wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5' or 3' end of the sense strand or antisense strand is positioned; ii) A second nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand and an antisense strand designed to reference different or the same gene to be silenced as described in i) above, wherein a single-stranded deoxyribonucleic acid (DNA) molecule is positioned conjugated to either the 5' or 3' end of the sense strand or the antisense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-stranded inhibitory ribonucleic acid (RNA) molecules.

[0010] In a preferred embodiment of the present invention, the single-stranded deoxyribonucleic acid (DNA) is linked to the first or second double-stranded inhibitory (RNA) molecule, and the linkage is i) the 5' sense strand of the first double-stranded inhibitory RNA and the 5' antisense stand of the second double-stranded RNA molecule, ii) The 5' sense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded RNA molecule, iii) The 5' sense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second inhibitory RNA molecule, iv) The 5' sense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, v) The 3' sense strand of the first double-stranded inhibitory RNA and the 5' sense strand of the second double-stranded inhibitory RNA molecule, vi) The 3' sense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded inhibitory RNA molecule, vii) The 3' sense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second double-stranded inhibitory RNA molecule, viii) The 3' sense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, ix) The 5' antisense strand of the first double-stranded inhibitory RNA and the 5' sense strand of the second double-stranded inhibitory RNA molecule, x) The 5' antisense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded inhibitory RNA molecule, xi) The 5' antisense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second double-stranded inhibitory RNA molecule, xii) The 5' antisense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, xiii) The 3' antisense strand of the first double-stranded inhibitory RNA and the 5' sense strand of the second double-stranded inhibitory RNA molecule, xiv) The 3' antisense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded inhibitory RNA molecule, xv) The 3' antisense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, xvi) Selected from the group consisting of the 3' antisense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second double-stranded inhibitory RNA molecule.

[0011] In a preferred embodiment of the present invention, the first and second genes to be silenced are the same gene.

[0012] In an alternative preferred embodiment of the present invention, the first and second genes to be silenced are different genes.

[0013] In a preferred embodiment of the present invention, the first and second double-stranded inhibitory RNA molecules contain different nucleotide sequences.

[0014] In a preferred embodiment of the present invention, the first or second gene to be silenced is the Apo B gene.

[0015] In a preferred embodiment of the present invention, the Apo B gene contains the nucleotide sequence shown in SEQ ID NO: 1, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

[0016] In a preferred embodiment of the present invention, the Apo B double-stranded inhibitory RNA contains, or consists of, a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 607 to 906.

[0017] In a preferred embodiment of the present invention, the Apo B double-stranded inhibitory RNA contains, or consists of, an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 907 to 1206.

[0018] In a further alternative preferred embodiment of the present invention, the first or second gene to be silenced is DGAT2.

[0019] In a preferred embodiment of the present invention, the DGAT2 gene contains the nucleotide sequence shown in SEQ ID NO: 6, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

[0020] In a preferred embodiment of the present invention, the double-stranded DGAT2 inhibitory RNA contains, or consists of, a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 to 306.

[0021] In a preferred embodiment of the present invention, the double-stranded DGAT2 inhibitory RNA contains, or consists of, an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 307 to 606.

[0022] In a preferred embodiment of the present invention, the double-stranded DGAT2 inhibitory RNA contains, or consists of, the antisense nucleotide sequence shown in SEQ ID NO: 4830.

[0023] In a preferred embodiment of the present invention, the double-stranded DGAT2 inhibitory RNA comprises or consists of the sense nucleotide sequence shown in SEQ ID NO: 4829.

[0024] In a preferred embodiment of the present invention, the double-stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4840 to 4848.

[0025] In a preferred embodiment of the present invention, the double-stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4831 to 4839.

[0026] In an alternative preferred embodiment of the present invention, the first or second gene to be silenced is PCSK9.

[0027] In a preferred embodiment of the present invention, the PCSK9 gene comprises the nucleotide sequence shown in SEQ ID NO: 2, and the double-stranded inhibitory RNA is 19 to 23 nucleotides in length.

[0028] In a preferred embodiment of the present invention, the double-stranded PCSK9 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 1207 to 1510.

[0029] In a preferred embodiment of the present invention, the double-stranded PCSK9 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 1511 to 1814.

[0030] In a preferred embodiment of the present invention, the double-stranded PCSK9 inhibitory RNA comprises or consists of the antisense nucleotide sequence shown in SEQ ID NO: 4822.

[0031] In a preferred embodiment of the present invention, the double-stranded PCSK9 inhibitory RNA comprises or consists of the sense nucleotide sequence shown in SEQ ID NO: 4821.

[0032] In a further alternative embodiment of the present invention, the first or second gene to be silenced is lipoprotein A (Lp(a)).

[0033] In a preferred embodiment of the present invention, the lipoprotein A gene comprises the nucleotide sequence described in Sequence ID No. 3, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

[0034] In a preferred embodiment of the present invention, the lipoprotein A double-strand inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 1815 to 2114.

[0035] In a preferred embodiment of the present invention, the lipoprotein A double-strand inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 2115 to 2414.

[0036] In a preferred embodiment of the present invention, the lipoprotein A double-strand inhibitory RNA comprises or consists of the antisense nucleotide sequence described in Sequence ID No. 4824.

[0037] In a preferred embodiment of the present invention, the lipoprotein A double-strand inhibitory RNA comprises or consists of the sense nucleotide sequence described in Sequence ID No. 4823.

[0038] In a further alternative embodiment of the present invention, the first or second gene to be silenced is angiotensinogen.

[0039] In a preferred embodiment of the present invention, the angiotensinogen gene comprises the nucleotide sequence shown in Sequence ID No. 4, and the double-stranded inhibitory RNA is 19 to 23 nucleotides long.

[0040] In a preferred embodiment of the present invention, the double-stranded angiotensinogen inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 2415 to 2714.

[0041] In a preferred embodiment of the present invention, the double-stranded angiotensinogen inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 2715 to 3014.

[0042] In a further alternative preferred embodiment of the present invention, the first or second gene to be silenced is Apo CIII.

[0043] In a preferred embodiment of the present invention, the Apo CIII gene comprises the nucleotide sequence shown in Sequence ID No. 5, and the double-stranded inhibitory RNA is 19 to 23 nucleotides long.

[0044] In a preferred embodiment of the present invention, the double-stranded ApoCIII inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3015 to 3314.

[0045] In a preferred embodiment of the present invention, the double-stranded ApoCIII inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3315 to 3614.

[0046] In a preferred embodiment of the present invention, the double-stranded ApoCIII inhibitory RNA comprises or consists of the sense nucleotide sequence shown in SEQ ID NO: 4860.

[0047] In a preferred embodiment of the present invention, the double-stranded ApoCIII inhibitory RNA comprises or consists of the antisense nucleotide sequence shown in Sequence ID No. 4859.

[0048] In a preferred embodiment of the present invention, the first or second gene to be silenced is ANGPTL3.

[0049] In a preferred embodiment of the present invention, the ANGPTL3 gene comprises the nucleotide sequence shown in Sequence ID No. 4819, and the double-stranded inhibitory RNA is 19 to 23 nucleotides long.

[0050] In a preferred embodiment of the present invention, the double-stranded ANGPTL3 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3615 to 3914.

[0051] In a preferred embodiment of the present invention, the double-stranded ANGPTL3 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3915 to 4214.

[0052] In a preferred embodiment of the present invention, the double-stranded ANGPTL3 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4826 or 4828.

[0053] In a preferred embodiment of the present invention, the double-stranded ANGPTL3 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4825 or 4827.

[0054] In a preferred embodiment of the present invention, the first or second gene to be silenced is ANGPTL4.

[0055] In a preferred embodiment of the present invention, the ANGPTL4 gene comprises the nucleotide sequence shown in SEQ ID NO: 4820, and the double-stranded inhibitory RNA is 19 to 23 nucleotides long.

[0056] In a preferred embodiment of the present invention, the ANGPTL4 double-stranded inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4215 to 4514.

[0057] In a preferred embodiment of the present invention, the ANGPTL4 double-stranded inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4515 to 4814.

[0058] In a preferred embodiment of the present invention, the first or second gene to be silenced is selected from the group consisting of: Apo B, DGAT2, PCSK9, Lp(a), APOCIII, angiotensinogen, ANGPTL3, and ANGPTL4.

[0059] In a preferred embodiment of the present invention, the first gene is Apo B, and the second gene is DGAT2.

[0060] In a preferred embodiment of the present invention, the first gene is Apo B, and the second gene is PCSK9.

[0061] In a preferred embodiment of the present invention, the first gene is Apo B, and the second gene is angiotensinogen.

[0062] In a preferred embodiment of the present invention, the first gene is Apo B, and the second gene is Apo CIII.

[0063] In a preferred embodiment of the present invention, the first gene is Apo B, and the second gene is Lp(a).

[0064] In a preferred embodiment of the present invention, the first gene is PCSK9, and the second gene is angiotensinogen.

[0065] In a preferred embodiment of the present invention, the first gene is PCSK9 and the second gene is Apo CIII.

[0066] In a preferred embodiment of the present invention, the first gene is PCSK9 and the second gene is DGAT2.

[0067] In a preferred embodiment of the present invention, the first gene is PCSK9 and the second gene is Lp(a).

[0068] In a preferred embodiment of the present invention, the first gene is angiotensinogen, and the second gene is Apo CIII.

[0069] In a preferred embodiment of the present invention, the first gene is angiotensinogen, and the second gene is DGAT2.

[0070] In a preferred embodiment of the present invention, the first gene is angiotensinogen, and the second gene is Lp(a).

[0071] In a preferred embodiment of the present invention, the first gene is Apo CIII, and the second gene is DGAT2.

[0072] In a preferred embodiment of the present invention, the first gene is Apo CIII, and the second gene is lipoproetin(a).

[0073] In a preferred embodiment of the present invention, the first gene is DGAT2, and the second gene is lipoprotein (a).

[0074] In a preferred embodiment of the present invention, the first gene is ANGPLT3 and the second gene is ApoB.

[0075] In a preferred embodiment of the present invention, the first gene is ANGPLT3 and the second gene is PCSK9.

[0076] In a preferred embodiment of the present invention, the first gene is ANGPLT3, and the second gene is angiotensinogen.

[0077] In a preferred embodiment of the present invention, the first gene is ANGPLT3 and the second gene is APOCIII.

[0078] In a preferred embodiment of the present invention, the first gene is ANGPLT3 and the second gene is Lp(a).

[0079] In a preferred embodiment of the present invention, the first gene is ANGPLT3 and the second gene is DGAT2.

[0080] In a preferred embodiment of the present invention, the first gene is ANGPLT3 and the second gene is ANPTLT4.

[0081] In a preferred embodiment of the present invention, the first gene is ANGPLT4 and the second gene is Apo B.

[0082] In a preferred embodiment of the present invention, the first gene is ANGPLT4 and the second gene is PCSK9.

[0083] In a preferred embodiment of the present invention, the first gene is ANGPLT4, and the second gene is angiotensinogen.

[0084] In a preferred embodiment of the present invention, the first gene is ANGPLT4 and the second gene is Apo CIII.

[0085] In a preferred embodiment of the present invention, the first gene is ANGPLT4 and the second gene is Lp(a).

[0086] In a preferred embodiment of the present invention, the first gene is ANGPLT4 and the second gene is DGAT2.

[0087] In an alternative preferred embodiment of the present invention, the first gene is DGAT2 and comprises the nucleotide sequence described in SEQ ID NO: 6, the double-stranded inhibitory RNA is 19 to 23 nucleotides long, and the double-stranded DGAT2 inhibitory RNA comprises or consists of the antisense nucleotide sequence described in SEQ ID NO: 4830 or 4840 to 4848, and / or the sense nucleotide sequence described in SEQ ID NO: 4829 or 4831 to 4839.

[0088] In a more preferred embodiment of the present invention, the first gene is PCSK9 and comprises the nucleotide sequence described in SEQ ID NO: 2, the double-stranded inhibitory RNA is 19 to 23 nucleotides long, and the double-stranded PCSK9 inhibitory RNA comprises or consists of the antisense nucleotide sequence described in SEQ ID NO: 4822 and / or comprises or consists of the sense nucleotide sequence described in SEQ ID NO: 4821.

[0089] In an alternative preferred embodiment of the present invention, the first gene is lipoprotein A and comprises the nucleotide sequence described in SEQ ID NO: 3, the double-stranded inhibitory RNA is 19 to 23 nucleotides in length, and the double-stranded lipoprotein A inhibitory RNA comprises or consists of the antisense nucleotide sequence described in SEQ ID NO: 4824 and / or the sense nucleotide sequence described in SEQ ID NO: 4823.

[0090] In a more preferred embodiment of the present invention, the first gene is ANGPTL3 and comprises the nucleotide sequence shown in SEQ ID NO: 4819, the double-stranded inhibitory RNA is 19 to 23 nucleotides long, and the double-stranded ANGPTL3 inhibitory RNA comprises or consists of the antisense nucleotide sequence shown in SEQ ID NO: 4826 or 4828, and / or comprises or consists of the sense nucleotide sequence shown in SEQ ID NO: 4825 or 4827.

[0091] In an alternative preferred embodiment of the present invention, the first gene is ApoC3 and comprises the nucleotide sequence described in SEQ ID NO: 5, the double-stranded inhibitory RNA is 19 to 23 nucleotides long, and the double-stranded ApoC3 inhibitory RNA comprises or consists of the antisense nucleotide sequence described in SEQ ID NO: 4859 and / or the sense nucleotide sequence described in SEQ ID NO: 4860.

[0092] In a preferred embodiment of the present invention, the single-stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815).

[0093] Preferably, the complementary single-stranded DNA contains the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816).

[0094] In a preferred embodiment of the present invention, the single-stranded DNA contains the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is bound to the 5' end of the sense nucleotide sequence.

[0095] In an alternative preferred embodiment of the present invention, the single-stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is bound to the 5' end of an antisense nucleotide sequence.

[0096] In a preferred embodiment of the present invention, the complementary single-stranded DNA contains the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is bound to the 5' end of the sense nucleotide sequence.

[0097] In a preferred embodiment of the present invention, the complementary single-stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is bound to the 5' end of the antisense nucleotide sequence.

[0098] In a preferred embodiment of the present invention, the first and / or second double-stranded inhibitory RNA molecule contains or consists of native nucleotides.

[0099] In alternative preferred embodiments of the present invention, the first and / or second double-strand inhibitory RNA molecule comprises modified nucleotides and / or modified sugars.

[0100] In preferred embodiments of the present invention, the modified nucleotide / sugar is a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a stereochemically restricted nucleotide, a restricted ethyl nucleotide, a non-basic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, The following are selected from the group: morpholinonucleotides, phosphoramidates, unnatural bases containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a phosphorodithioate (PS2), nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimetic compounds, such as 5'-vinyl phosphate, 2'-deoxy-2'-fluoro, and nucleotides containing 2'-methyl sugar bases and glycol nucleic acids.

[0101] In a preferred embodiment of the present invention, the first or second double-stranded inhibitory RNA molecule comprises at least one modified nucleotide, wherein the modification is 2'-deoxy-2'-fluoro.

[0102] In a preferred embodiment of the present invention, the first and / or second double-stranded inhibitory RNA molecule comprises at least one modified nucleotide, wherein the modification is 2'-O-methyl.

[0103] In a more preferred embodiment of the present invention, the first and / or second double-stranded inhibitory RNA molecule comprises at least one phosphorothioate bond.

[0104] In a more preferred embodiment of the present invention, the first and / or second double-stranded inhibitory RNA molecule comprises at least one 5'-vinyl phosphate.

[0105] In one embodiment of the present invention, the first and / or second double-stranded inhibitory RNA molecule contains at least one modified sugar.

[0106] Sugar modification includes modified versions of the ribosyl moiety, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, e.g., 2'-O-methyl, T-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, -O-propargyl, 2'-O-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl), etc. -O-modified RNA; 2'-deoxy(DNA); e.g., 2'-O-(2-chloroethoxy)methyl (M Examples include 2'-O-(haloalkoxy)methyl compounds such as CEM, -O-(2,2-dichloroethoxy)methyl (DCEM); 2'-<3-alkoxycarbonyl compounds such as T-O-[2-(methoxycarbonyl)ethyl](MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl](MCE), and T-O-[2-(N,N-dimethylcarbamoyl)ethyl](DCME); modifications of 2'-halo, carbasaccharides, and azasaccharides such as 2'-F, FANA (2'-F arabinosyl nucleic acid); and 3'-O-alkyl compounds such as 3'-O-methyl, 3'-O-butyryl, and VO-propargyl, and their derivatives.

[0107] In a preferred embodiment of the present invention, a nucleic acid molecule according to the present invention is provided, which comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

[0108] In a preferred embodiment of the present invention, a nucleic acid molecule according to the present invention is provided, which comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4821) and an antisense strand (SEQ ID NO: 4822), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

[0109] In a preferred embodiment of the present invention, a nucleic acid molecule according to the present invention is provided, which comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4823) and an antisense strand (SEQ ID NO: 4824), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

[0110] In a preferred embodiment of the present invention, a nucleic acid molecule according to the present invention is provided, which comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

[0111] In a preferred embodiment of the present invention, a nucleic acid molecule according to the present invention is provided, which comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4827) and an antisense strand (SEQ ID NO: 4828), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

[0112] In a preferred embodiment of the present invention, a nucleic acid molecule according to the present invention is provided, which comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4860) and an antisense strand (SEQ ID NO: 4859), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

[0113] In a preferred embodiment of the present invention, i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4821) and an antisense strand (SEQ ID NO: 4822), wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A nucleic acid molecule comprising a second nucleic acid comprising a double-strand inhibitory (RNA) molecule including a sense strand (SEQ ID NO: 4823) and an antisense strand (SEQ ID NO: 4824), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-strand inhibitory (RNA) molecules.

[0114] In a preferred embodiment of the present invention, i) A first nucleic acid comprising a double-strand inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826), wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A second nucleic acid comprising a double-strand inhibitory (RNA) molecule including a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-strand inhibitory (RNA) molecules.

[0115] In a preferred embodiment of the present invention, i) A first nucleic acid comprising a double-strand inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830), wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A second nucleic acid comprising a double-strand inhibitory (RNA) molecule including a sense strand (SEQ ID NO: 4860) and an antisense strand (SEQ ID NO: 4859), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-strand inhibitory (RNA) molecules.

[0116] In a preferred embodiment of the present invention, the single-stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815).

[0117] Preferably, the complementary single-stranded DNA contains the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816).

[0118] In a preferred embodiment of the present invention, the single-stranded DNA contains the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is bound to the 5' end of the sense nucleotide sequence.

[0119] In an alternative preferred embodiment of the present invention, the single-stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is bound to the 5' end of an antisense nucleotide sequence.

[0120] In a preferred embodiment of the present invention, the single-stranded DNA contains the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is bound to the 5' end of the sense nucleotide sequence.

[0121] In a preferred embodiment of the present invention, the single-stranded DNA contains the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is bound to the 5' end of an antisense nucleotide sequence.

[0122] In a more preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4849 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4822.

[0123] In an alternative, further preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4850 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4822.

[0124] In a more preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4851 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4824.

[0125] In a more preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4852, and an antisense strand comprising or comprising the nucleotide sequence shown in SEQ ID NO: 4824.

[0126] In a more preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4853 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4826.

[0127] In an alternative, further preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4854 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4826.

[0128] In a more preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4855, and an antisense strand comprising or comprising the nucleotide sequence shown in SEQ ID NO: 4828.

[0129] In an alternative and more preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4856 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4828.

[0130] In an alternative, further preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4857 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4830.

[0131] In an alternative, further preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4858 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4830.

[0132] In an alternative, further preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4861 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4859.

[0133] In an alternative, further preferred embodiment of the present invention, the nucleic acid comprises or comprises a sense strand comprising the nucleotide sequence shown in SEQ ID NO: 4862 and an antisense strand comprising the nucleotide sequence shown in SEQ ID NO: 4859.

[0134] In a preferred embodiment of the present invention, i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand selected from the group consisting of SEQ ID NOs. 607-906 and an antisense strand selected from the group consisting of SEQ ID NOs. 907-1206, wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A second nucleic acid molecule comprising a double-strand inhibitory ribonucleic acid (RNA) molecule, the second nucleic acid molecule comprising a sense strand selected from the group consisting of SEQ ID NOs. 7-306, 4829, 4831-4839, 4857 and 4858, and an antisense strand selected from the group consisting of SEQ ID NOs. 307-606, 4830 and 4840-4848, wherein a single-strand deoxyribonucleic acid (DNA) molecule is positioned conjugated to the 5' of the sense strand, the single-strand deoxyribonucleic acid (DNA) molecule being substantially complementary to the single-strand deoxyribonucleic acid (DNA) molecule described in i) above, and annealing by complementary base pairing to form a double-strand DNA linker that links the first and second double-strand inhibitory ribonucleic acid (RNA) molecules.

[0135] In a preferred embodiment of the present invention, the second nucleic acid comprises a double-strand inhibitory (RNA) molecule having a sense strand represented by SEQ ID NO: 4857 and an antisense strand represented by SEQ ID NO: 4830.

[0136] In an alternative preferred embodiment of the present invention, the second nucleic acid comprises a double-strand inhibitory (RNA) molecule comprising a sense strand shown in SEQ ID NO: 4858 and an antisense strand shown in SEQ ID NO: 4830.

[0137] In a preferred embodiment of the present invention, the nucleic acid molecule is covalently bonded to N-acetylgalactosamine.

[0138] The sugar moiety in N-acetylgalactosamine may contain glycosidic bonds to improve stability. Various glycosidic bonds are known in the art and are formed between the hemiacetal of the sugar moiety and several chemical groups that form O-, N-, S-, or C-glycosidic bonds. N-acetylgalactosamine preferably contains O-, N-, S-, or C-glycosidic bonds.

[0139] In a further embodiment of the present invention, the N-acetylgalactosamine is ligated to either the antisense portion or the sense portion of the inhibitory RNA.

[0140] In a further embodiment of the present invention, the N-acetylgalactosamine is ligated to either the antisense strand or the sense strand of the double-stranded inhibitory RNA molecule of the first nucleic acid.

[0141] In a further embodiment of the present invention, the N-acetylgalactosamine is ligated to either the antisense strand or the sense strand of the double-stranded inhibitory RNA molecule of the second nucleic acid.

[0142] In a further embodiment of the present invention, N-acetylgalactosamine is ligated to either the antisense strand or the sense strand of the double-strand inhibitory RNA molecule of the first nucleic acid, and ligated to either the antisense strand or the sense strand of the double-strand inhibitory RNA molecule of the second nucleic acid.

[0143] Preferably, N-acetylgalactosamine is ligated to the 3' end of the sense RNA.

[0144] In an alternative embodiment of the present invention, N-acetylgalactosamine is ligated to the 5' end of the sense RNA.

[0145] In an alternative preferred embodiment of the present invention, the N-acetylgalactosamine is ligated to the 3' end of the antisense RNA.

[0146] In a preferred embodiment of the present invention, N-acetylgalactosamine is monovalent.

[0147] In a preferred embodiment of the present invention, N-acetylgalactosamine is divalent.

[0148] In an alternative embodiment of the present invention, N-acetylgalactosamine is trivalent.

[0149] In a preferred embodiment of the present invention, the nucleic acid molecule is covalently bonded to a molecule having the following structure:

[0150] [ka]

[0151] In an alternative embodiment of the present invention, the nucleic acid molecule is covalently bonded to a molecule having the following structure:

[0152] [ka]

[0153] In an alternative embodiment of the present invention, the nucleic acid molecule is covalently bonded to a molecule having the following structure:

[0154] [ka]

[0155] In an alternative embodiment of the present invention, the nucleic acid molecule is covalently bonded to a molecule having the following structure:

[0156] [ka]

[0157] In an alternative embodiment of the present invention, the nucleic acid molecule is covalently bonded to a molecule having the following structure:

[0158] [ka]

[0159] In an alternative embodiment of the present invention, the nucleic acid molecule is covalently bonded to a molecule having the following structure:

[0160] [ka]

[0161] A further aspect of the present invention provides a drug comprising nucleic acid according to the present invention.

[0162] A further aspect of the present invention provides a pharmaceutical composition comprising nucleic acid molecules according to the present invention.

[0163] In a preferred embodiment of the present invention, the composition further comprises a pharmaceutical carrier and / or an excipient.

[0164] A further aspect of the present invention provides nucleic acid molecules or pharmaceutical compositions according to the present invention for use in the treatment or prevention of subjects having or being predisposed to hypercholesterolemia.

[0165] In preferred embodiments of the present invention, its use is for the treatment or prevention of diseases associated with hypercholesterolemia.

[0166] In preferred embodiments of the present invention, the diseases associated with hypercholesterolemia are selected from the group consisting of stroke prevention, hyperlipidemia, cardiovascular disease, atherosclerosis, coronary heart disease, molar arch, cerebrovascular disease, peripheral artery disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Berger's disease, renal artery stenosis, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis.

[0167] When administered, the compositions of the present invention are administered as pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents such as cholesterol-lowering agents, which can be administered separately from the nucleic acid molecules of the present invention, or in combination preparations where the combination is compatible.

[0168] The nucleic acid according to the present invention, in combination with other different therapeutic agents, is administered simultaneously, sequentially, or as separate doses over time.

[0169] The therapeutic agent of the present invention can be administered by any conventional route, including injection, or by gradual infusion over time. Administration may be, for example, orally, intravenously, intraperitoneally, intramuscularly, intracavitarially, subcutaneously, percutaneously, or perepithelially.

[0170] The compositions of the present invention are administered in an effective dose. An effective dose is the amount of the composition that produces a desired response, either alone or in combination with additional doses. When treating diseases such as cardiovascular disease, the desired response is to inhibit or reverse the progression of the disease. This may involve only a temporary slowing of disease progression, but more preferably a permanent cessation of disease progression. This can be monitored by routine means.

[0171] These amounts naturally depend on individual patient parameters, including the specific condition being treated, the severity of the condition, age, physical condition, size, and weight, the duration of treatment, the nature of concomitant therapies (if any), the specific route of administration, and similar factors within the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed solely through routine experimentation. It is generally preferable to use the maximum dose of each component or combination, i.e., the safest dose according to sound medical judgment. However, those skilled in the art will understand that patients may request lower doses or tolerable doses for medical, psychological, or factually other reasons.

[0172] The pharmaceutical composition used in the aforementioned method is preferably sterile and contains an effective amount of the nucleic acid molecule according to the present invention to produce a desired response in a unit of weight or volume suitable for administration to a patient. The response may be measured, for example, by determining cardiovascular disease regression and reduction of disease symptoms.

[0173] The dose of the nucleic acid molecule according to the present invention administered to a subject may be selected according to different parameters, particularly the mode of administration used and the condition of the subject. Other factors include the desired duration of treatment. If the response in the subject is insufficient with the initial dose applied, a higher dose (or an effectively higher dose via a different, more localized delivery route) may be employed, as long as the patient's tolerance allows. It will be evident that the nucleic acid detection method according to the present invention facilitates the determination of an appropriate dosage for subjects requiring treatment.

[0174] Generally, doses of nucleic acid molecules disclosed herein, ranging from 1 nM to 1 μM, are generally formulated and administered according to standard procedures. Preferably, doses may range from 1 nM to 500 nM, 5 nM to 200 nM, or 10 nM to 100 nM. Those skilled in the art will be aware of other protocols for the administration of compositions with different dosages, injection schedules, injection sites, and modes of administration than those described above. Administration of compositions to non-human mammals (e.g., for experimental or veterinary therapeutic purposes) is carried out under substantially the same conditions as described above. Subjects, as used in this disclosure, are mammals, preferably humans, and include non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents.

[0175] When administered, the pharmaceutical preparations of the present invention are applied in pharmaceutically acceptable amounts and compositions. The term "pharmaceutically acceptable" means non-toxic materials that do not interfere with the efficacy of the biological activity of the active ingredient. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents, such as statins. When used in pharmaceuticals, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be used for convenience in preparing such pharmaceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacokinetic and pharmaceutically acceptable salts include, but are not limited to, salts prepared from acids such as hydrochloric acid, hydrobromide, sulfates, nitrates, phosphates, maleates, acetates, salicylates, citrates, formates, malons, and succinates. Furthermore, pharmaceutically acceptable salts may be prepared as alkali metal salts or alkaline earth salts, such as sodium salts, potassium salts, or calcium salts.

[0176] The composition may be combined with a pharmaceutically acceptable carrier, if desired. As used in this disclosure, the term "pharmaceutically acceptable carrier" means one or more suitable solid or liquid fillers, diluents, or encapsulants suitable for administration to humans. In this context, the term "pharmaceutically acceptable carrier" refers, for example, to natural or synthetic organic or inorganic components to which the active ingredient is combined to enhance solubility and / or stability. Components of the pharmaceutical composition may also resonate with and with the molecules of the present invention, such that there are no interactions that substantially impair the desired pharmaceutically effectiveness.

[0177] The pharmaceutical composition may also contain a suitable buffer, including acetic acid in the salt, citric acid in the salt, boric acid in the salt, and phosphoric acid in the salt. The pharmaceutical composition may also optionally contain a suitable preservative.

[0178] Pharmaceutical compositions may be conveniently presented in unit dosage forms and may be prepared by any method well known in the pharmaceutical art. All of these methods involve associating an active agent with a carrier constituting one or more minor components. Generally, compositions are prepared by homogeneously and closely associating an active compound with a liquid carrier, a micronized solid carrier, or both, and then, if necessary, shaping the product.

[0179] A composition suitable for parenteral administration preferably comprises a sterile aqueous or non-aqueous preparation of nucleic acid, which is preferably isotonic with the recipient's blood. This preparation may be formulated according to known methods using suitable dispersants or wetting agents and suspension agents. The sterile injection preparation may also be a sterile injection solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixative oils are those conventionally used as solvents or suspension media. For this purpose, any non-irritating fixative oil containing synthetic monoglycerides or diglycerides may be used. In addition, fatty acids such as oleic acid may be used in the preparation of injections. Carrier formulations suitable for oral, subcutaneous, intravenous, and intramuscular administration are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0180] A further aspect of the present invention provides a method for treating a subject having or being predisposed to hypercholesterolemia, comprising administering an effective amount of a nucleic acid or pharmaceutical composition according to the present invention to treat or prevent hypercholesterolemia.

[0181] In the preferred method of the present invention, the hypercholesterolemia is familial hypercholesterolemia.

[0182] A preferred method of the present invention provides treatment or prevention of diseases associated with hypercholesterolemia.

[0183] In the preferred method of the present invention, the diseases associated with hypercholesterolemia are selected from the group consisting of stroke prevention, hyperlipidemia, cardiovascular disease, atherosclerosis, coronary heart disease, molar and dental arch disease, cerebrovascular disease, peripheral artery disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Berger's disease, renal artery stenosis, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis.

[0184] Throughout this specification and the claims, the words “comprise” and “contain,” and variations thereof such as “comprising” and “comprises,” mean “including, but not limited to,” and are not intended to exclude (or exclude) other parts, additives, components, integers, or steps. “Essentially consisting of” means having essential elements but including elements that do not substantially affect the function of those essential elements.

[0185] Throughout this specification and the claims, the singular form includes the plural form unless the context requires otherwise. In particular, where the article is used, this specification should be understood to intend both singular and plural singular forms unless the context requires otherwise.

[0186] [Brief explanation of the drawing]

[0187] It should be understood that any configurations, elements, features, compounds, chemical parts, or groups described in relation to a particular aspect, embodiment, or example of the present invention are applicable to any other aspect, embodiment, or example described herein, provided that they are not incompatible.

[0188] Embodiments of the present invention will be described here only illustratively, with reference to the following materials, methods, and examples. [Examples]

[0189] material and method Transfection in primary mouse hepatocytes Double-stranded siRNAs and heterodimerized siRNAs (Tables 3 and 5) synthesized by Bio-Synthesis (Louisville, Texas) were resuspended in nuclease-free water (Invitrogen® AM9932) to produce 10 μM stock solutions. For serum stability assays, stock siRNAs were incubated at 37°C for 2 hours in a vehicle (nuclease-free water) or in human serum (HS) at various concentrations (20%–80%). After pre-incubation in serum or vehicle, siRNAs were transfected into primary mouse hepatocytes in triplicate in 384-well plates (Thermo Scientific® 164688) at various concentrations ranging from 2.5 nM to 100 nM using 0.15 μL of lipofectamine RNAiMAX (Invitrogen® 13778075) per well. Transfected cells were incubated at 37°C and 5% CO2 for 48 hours. Cells that had not been treated with siRNA were used as a control.

[0190] GalNAc-conjugate ApoB-mTTR heterodimer free uptake assay in primary mouse hepatocytes Standard siRNA controls (mTTR and ApoB_C3_01) and GalNAc-conjugate ApoB-mTTR heterodimers (Table 7) were synthesized using Bio-Synthesis (Lewisville, TX). The GalNAc-conjugate constructs and standard siRNA controls were resuspended in nuclease-free water (Invitrogen® AM9932) to produce 10 μM stock solutions. The stock siRNAs were partitioned three times in a 384-well plate (Thermo Scientific® 164688) to final concentrations of 4 nM, 25 nM, and 100 nM, and primary mouse hepatocytes were added at a concentration of 5,000 cells per well for the free uptake assay. After treatment, cells were incubated at 37°C and 5% CO2 for 48 hours. Cells not treated with siRNA were used as a control.

[0191] Duplex RT-qPCR Cells were processed for RT-qPCR readout using the Cells-to-CT 1-Step TaqMan Kit (Invitrogen® A25603). Briefly, cells were washed with 50 μL of ice-cold PBS and lysed in 20 μL of lysis solution containing DNase I. Lysis was stopped for 2 minutes after 5 minutes by adding 2 μL of stop solution. For RT-qPCR analysis, 1 μL of lysate per well was dispensed into a 96-well PCR plate in a 10 μL RT-qPCR reaction volume. RT-qPCR was performed using the TaqMan® 1-step qRT-PCR mix from the Cells-to-CT 1-Step TaqMan kit, with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1), or DGAT2 (FAM, Assay Id Mm00499536_m1). RT-qPCR was performed using a QuantStudio 5 thermocycling instrument (Applied BioSystems). Relative quantification was determined using the ΔΔT method (without siRNA treatment), with GAPDH used as an internal control and expression changes normalized to the reference sample.

[0192] Receptor-mediated uptake assay between GalNAc-conjugated ApoB-DGAT2 heterodimer and single siRNA control GalNAc-conjugated ApoB (CS13) and DAGT2 (CS8) single siRNA controls, as well as ApoB-DGAT2 (CS15 and CS16) heterodimer siRNAs (Table 3), were synthesized using CatSci (Cardiff, UK). The constructs were resuspended in nuclease-free water (Invitrogen® AM9932) to prepare 100 μM stock solutions.

[0193] Newly prepared, or *Stock siRNA, pre-incubated in serum, was dispensed three times into collagen-coated 384-well plates (Corning® 354666) to final concentrations of 0.1 nM, 1 nM, 4 nM, and 25 nM. Primary mouse hepatocytes were added at a concentration of 6,000 cells per well. After treatment, the cells were incubated at 37°C and 5% CO2 for 48 hours. Cells not treated with siRNA were used as a control. (*Stock siRNA was exposed to 80% human serum (HS) at 37°C for 2 hours).

[0194] Duplex RT-qPCR Cells were processed for RT-qPCR readout using the Cells-to-CT 1-Step TaqMan Kit (Invitrogen® A25603). Briefly, cells were washed with 50 μL of ice-cold PBS and lysed in 20 μL of lysis solution containing DNase I. Lysis was stopped after 5 minutes by adding 2 μL of stop solution for 2 minutes. For RT-qPCR analysis, 1 μL of lysate per well was dispensed into a 96-well PCR plate with a total RT-qPCR reaction volume of 10 μL. RT-qPCR was performed using the TaqMan® 1-step qRT-PCR mix from the Cells-to-CT 1-Step TaqMan kit, with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1), or DGAT2 (FAM, Assay Id Mm00499536_m1). RT-qPCR was performed using a QuantStudio 5 thermocycling instrument (Applied BioSystems). Relative quantification was determined using the ΔΔCT method, with GAPDH used as an internal control and expression changes normalized to the reference sample (without siRNA treatment).

[0195] In vivo mouse studies method Male C57BL / 6J mice (20–25 g) were housed in groups in the Saretius Animal Unit at the University of Reading, maintained at 23°C under a 12-hour light-dark cycle with humidity controlled according to home office regulations. The mice were given access to standard rodent solid feed SDS rat growth diet (RM3-E-FG) throughout the study period.

[0196] Formulation of siRNA compounds Heterodimeric compounds CS15 and CS16 (Table 3) were formulated in RNase-free PBS at concentrations of 2 and 5 mg / mL, respectively, to provide doses of 10 and 20 mg / kg when administered subcutaneously (SC) at a dose volume of 5 mL / kg. Single siRNA control compounds CS8, CS13, and PAR-ApoB control (Table 3) were formulated in RNAase-free PBS to concentrations of 1 and 2 mg / mL, respectively, to obtain doses of 5 and 10 mg / kg when administered SC at a dose volume of 5 mL / kg. The mTTR control siRNA (Table 3) was formulated in RNAase-free PBS to a concentration of 2 mg / mL to provide a dose of 10 mg / kg when administered SC at a dose volume of 5 mL / kg.

[0197] Liver preparation for RT-qPCR On days 5 and 14 (n=4) after injection of siRNA compound or vehicle, each treatment group was finally sampled by cardiac puncture under isoflurane. Liver tissue was excised and rapidly frozen in liquid N2. Total RNA was extracted from rapidly frozen homogenates of the entire liver using the QIAGEN RNeasy Mini Kit (74104).

[0198] Duplex RT-qPCR was performed using ThermoFisher TaqMan Fast 1-Step Master Mix with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1), or DGAT2 (FAM, Assay Id Mm00499536_m1). Relative quantification (RQ) of target mRNA was determined using the ΔΔCT method, and changes in target gene expression were normalized to the vehicle control using GAPDH as an internal control.

[0199] [Table 1] TIFF2026518570000008.tif63160

[0200] Example 1 As shown in Table 1, when transfected into primary mouse hepatocytes at concentrations of 2.5–100 nM to test efficacy, ApoB_C3_S19 and ApoB_C3_S18 (single siRNAs with complementary DNA hybridized to the 5' end of the sense strand – Table 3) lacked complementary DNA hybridized to a DNA crack at the 5' end of the sense strand and showed ApoB knockdown comparable to ApoB_C3_OR. Regarding the ApoB-mTTR heterodimer, the mTTR moiety showed mTTR target knockdown comparable to modified single siRNA mTTR (bottom two columns of Table 1), while the ApoB moiety showed reduced efficacy (KD range: 64.2%–76.3%) compared to the single siRNA counterpart ApoB_C3_OR (KD range: 90.1%–92.8%).

[0201] To test stability in human serum, all siRNA constructs were pre-incubated in human serum for 2 hours and transfected in primary mouse hepatocytes for 48 hours when target gene knockdown was measured by RT-qPCR. As shown in Table 2, the mTTR portion of the ApoB-mTTR heterodimer was stable across different concentrations of human serum, exhibiting KD levels of 87%–94.5%, which was comparable to the KD levels of standard mTTR siRNA (74.3%–93.1% KD). For the ApoB portion of the ApoB-mTTR heterodimer siRNA, serum stability was high in 20% human serum, showing KD levels of 92.1% and 84.7% at 2.5 nM and 25 nM, respectively. KD decreased after incubation in 80% human serum, showing KD values ​​of 32.5% and 23.9% at 2.5 nM and 25 nM, respectively. In contrast, the standard ApoB siRNA counterpart, ApoB_C3_OR, showed stable KD across different serum concentrations ranging from 82.4% to 97.6% (Table 2).

[0202] [Table 2]

[0203] [Table 3]

[0204] [Table 4]

[0205] Example 2 As shown in Table 4, DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers pre-incubated in a vehicle showed high levels of knockdown (KD) against both target genes DGAT2 and ApoB when transfected into primary mouse hepatocytes at a concentration of 25 nM (KD range: 82.7%–93%). When pre-incubated in 50% human serum and subsequently transfected into primary mouse hepatocytes at 25 nM, both DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers showed high KD levels completely equivalent to those obtained with pre-incubation in a vehicle (KD range after serum pre-incubation at 25 nM: 75.9%–89.8%), suggesting stability in 50% human serum. Interestingly, the KD levels of the target genes ApoB and DGAT2 were demonstrated by DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers when pre-incubated in vehicle or 50% human serum, followed by transfection, and were completely equivalent in KD levels at 25 nM to their single siRNA control counterparts, ApoB_C3_OR, ApoB_REVCR, and DGAT2_3 (siRNA control KD range: 90.2%–93.7%). A complete description of the siRNA structures is shown in Table 5.

[0206] Regarding the GS-mTTR-A and GS-mTTR-B heterodimers, pre-incubation in a vehicle, when transfected into primary mouse hepatocytes at a concentration of 25 nM, resulted in high levels of KD (KD range: 83.2%–97%) for both target genes ApoB and mTTR. Pre-incubation in 50% human serum followed by transfection in primary mouse hepatocytes at 25 nM resulted in both GS-mTTR-A and GS-mTTR-B heterodimers exhibiting high KD levels completely equivalent to those achieved by pre-incubation in a vehicle (KD range after serum pre-incubation, followed by transfection at 25 nM: 87.5%–96.2%). This suggests high serum stability for these heterodimers. The KD levels of the target genes ApoB and mTTR were shown by GS-mTTR-A and GS-mTTR-B heterodimers when transfected after pre-incubation with vehicle or 50% human serum, and were completely equivalent to the KD levels of their single siRNA control counterparts mTTR, mTTR-AC, mTTR-ARG, PAR-ApoB, GS-AC, and GS-ARG (siRNA control KD range: 90.2%–98.2%). A complete description of the siRNA structures used here can be found in Table 5.

[0207] [Table 5]

[0208] [Table 6] TIFF2026518570000014.tif110161

[0209] Example 3 As shown in Table 6, the ApoB-mTTR_AG1 and ApoB-mTTR_AG2 heterodimers exhibited ApoB KD levels of 51.9% and 57%, respectively, at 100 nM and 48% and 46.2%, respectively, at 25 nM, in free uptake. When primary hepatocytes were treated with the single ApoB siRNA counterpart ApoB_C3_01 at 100 nM and 25 nM, the KD levels were 68.6% and 54.5%, respectively, suggesting that the heterodimers functioned with nearly the same efficiency as the single siRNA control in silencing the target gene ApoB. Overall, the ApoB-mTTR heterodimer structures demonstrated high effectiveness in silencing mTTR targets in all ApoB-mTTR heterodimers tested herein, exhibiting mTTR KD levels ranging from 88.4% to 97.7% with 25 nM and 100 nM treatments in free uptake assays. The mTTR KD values ​​exhibited by treatment with the heterodimers were similar to those obtained with single mTTR siRNA control treatments (KD levels at 25 nM and 100 nM: 98.1%). A complete description of the siRNA structures used is shown in Table 7.

[0210] [Table 7]

[0211] [Table 8] TIFF2026518570000017.tif60167

[0212] Example 4 As shown in Table 8, ApoB-DGAT2 (CS15 and CS16) heterodimerized siRNAs exhibited 64% and 60% ApoB KD levels at 25 nM and 49% and 50% KD levels at 4 nM, respectively, upon receptor-mediated uptake in primary mouse hepatocytes. When cells were treated with the single siRNA counterpart CS13, ApoB KD was 72% and 53% at 25 nM and 4 nM, respectively. For DGAT2 target KD, the heterodimerized siRNAs exhibited KD levels of 76% and 65% at 25 nM and 55% and 57% at 4 nM. In comparison, single siRNA CS8 yielded 76% DGAT2 KD at 25 nM and 48% DGAT2 KD at 4 nM.

[0213] These results demonstrate that, in receptor-mediated assays, ApoB-DGAT2 heterodimerized siRNA functions similarly to a single ApoB or DGAT2 siRNA of the same sequence, resulting in highly effective silencing of two targets in the same cell. A complete description of the siRNA structures used can be found in Table 9.

[0214] [Table 9]

[0215] Example 5 To test stability in human serum, siRNA constructs were pre-incubated in 80% human serum (HS) at 37°C for 2 hours, followed by receptor-mediated uptake assays in primary mouse hepatocytes. Knockdown of the target gene was measured 48 hours later by RT-qPCR.

[0216] As shown in Table 8, the ApoB-DGAT2 heterodimerized siRNAs CS15 and CS16 exhibited comparable levels of target KD compared to single siRNAs of the same sequence, CS13 and CS8. ApoB KD for the CS15 and CS16 heterodimerized siRNAs was 71% and 73% at 25 nm, and 52% and 49% at 4 nM. This silencing level is preferable to that shown by the single siRNA counterpart CS13, which has KDs of 71% and 60% at 25 nM and 4 nM, respectively. Similarly, the DGAT2 KD levels achieved by the heterodimerized siRNAs were 75% and 73% at 25 nM (64% and 58% at 4 nM), compared to 80% and 67% at 25 nM and 4 nM for the single siRNA CS8, respectively.

[0217] These results demonstrate that the ApoB-DGAT2 heterodimerized siRNA is highly stable in 80% human serum and functions similarly to a single siRNA of the same sequence in receptor-mediated uptake assays. A complete description of the siRNA structure used can be found in Table 9 below.

[0218] [Table 10]

[0219] Example 4 In vivo silencing effect of heterodimer siRNA (ApoB-DGAT2) compared to single siRNA control. Mouse test Four mice in each treatment group were subcutaneously injected (SC) with either a vehicle (PBS), a GalNAc-conjugated ApoB-DGAT2 (CS15 or CS16) heterodimer, or an ApoB (CS13) or DAGT2 (CS8) single siRNA control (Table 2). Positive siRNA controls included PAR-ApoB and mTTR (Table 2).

[0220] Each compound was administered to heterodimerized siRNA at either 5 mg / kg or 10 mg / kg (single siRNA), corresponding to 10 mg / kg and 20 mg / kg respectively. After sacrificing on either day 5 or day 14, liver target mRNA levels were measured by RT-qPCR, and the knockdown % (KD) was determined compared to the vehicle-treated control.

[0221] Table 10 shows in vivo silencing of liver target mRNAs (ApoB and DGAT2) by heterodimerized siRNA compounds (CS15 and CS16) compared to single ApoB or DGAT2 siRNA controls (CS13 and CS8, respectively).

[0222] The CS15 heterodimer (20 mg / kg) yielded 53% KD for both ApoB and DGAT2 mRNA on day 5, while the CS16 heterodimer yielded 62% and 54% KD for ApoB and DGAT2, respectively. Single siRNAs CS13 and CS8 at equivalent doses (10 mg / kg) yielded 61% and 49% KD for ApoB and DGAT2, respectively. A positive control siRNA (PAR-ApoB;Alnylam) was similarly performed, yielding 63% ApoB KD on day 5. ApoB silencing decreased on day 14 for the heterodimer CS15 and single siRNA CS13 (13% and 11%, respectively), which was not unexpected considering that the positive control of the same sequence (PAR-ApoB;Alnylam) performed similarly (17% KD). However, the heterodimer CS16 provided 34% KD at D14.

[0223] In contrast, DGAT2 mRNA silencing was maintained in mice treated with either a Day 14 heterodimer (CS15 67% KD, CS16 57%) or a single siRNA CS8 (51%). Importantly, there was a significant increase in DGAT2 silencing with a single DGAT2 siRNA CS15 heterodimer (Day 14) (P=0.02 low dose, P=0.008 high dose). For CS16 heterodimers at low doses of CS8, ApoB knockdown on Day 14 was significantly greater compared to single ApoB siRNA CS13 (P=0.04).

[0224] Statistical analysis performed: Two-way Anova and Tukey post-hoc tests These results demonstrate that both ApoB and DGAT2-targeted KD (%) in vivo with heterodimerized siRNA is equivalent to or superior to single siRNA controls at both low (5 mg / kg) and high (10 mg / kg) doses, and at both time points (day 5 and day 14).

[0225] [Table 11]

[0226] Table 11 shows the comparative efficacy (%) of each single siRNA (ApoB-DGAT2 heterodimer siRNA) on day 5 and day 14. [Percentage of KD by heterodimer siRNA (ApoB or DGAT2) divided by percentage of KD by monosiRNA (x100)].

[0227] Equivalent or superior performance is achieved with heterodimerized siRNA compared to a single siRNA control. At low doses, for ApoB KD, heterodimers showed efficacy of 134–164% (day 5) and 210–280% (day 14), while at high doses, they showed efficacy of 87–102% (day 5) and 114–298% (day 14). For DGAT2 KD, comparative efficacy was 84–124% (day 5) and 102–147% (day 14) at lower doses, while at higher doses it was 109–111% (day 5) and 112–131% (day 14).

[0228] [Table 12]

[0229] Example 5: Alternative Sequence

[0230] [Table 13]

[0231] [Table 14] TIFF2026518570000024.tif116164

[0232] [Table 15] a, c, g, i, and u represent 2'-O-methyladenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverse basic deoxyribose residue; and (NAG37) indicates the following:

[0233] [ka]

Claims

1. nucleic acid molecules, i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand designed with reference to a nucleotide sequence containing a gene to be silenced, and an antisense strand, wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5' or 3' end of the sense strand or antisense strand is positioned; ii) A second nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand and an antisense strand designed to reference different or the same gene to be silenced as described in i) above, wherein a single-stranded deoxyribonucleic acid (DNA) molecule is positioned conjugated to either the 5' or 3' end of the sense strand or the antisense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-stranded inhibitory ribonucleic acid (RNA) molecules.

2. A nucleic acid molecule according to claim 1, wherein the single-stranded deoxyribonucleic acid (DNA) is linked to the first or second double-stranded inhibitory (RNA) molecule, and the linkage is i) The 5' sense strand of the first double-stranded inhibitory RNA and the 5' antisense stand of the second double-stranded RNA molecule, ii) The 5' sense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded RNA molecule, iii) The 5' sense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second inhibitory RNA molecule, iv) The 5' sense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, v) The 3' sense strand of the first double-stranded inhibitory RNA and the 5' sense strand of the second double-stranded inhibitory RNA molecule, vi) The 3' sense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded inhibitory RNA molecule, vii) The 3' sense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second double-stranded inhibitory RNA molecule, viiii) The 3' sense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, ix) The 5' antisense strand of the first double-stranded inhibitory RNA and the 5' sense strand of the second double-stranded inhibitory RNA molecule, x) The 5' antisense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded inhibitory RNA molecule, xi) The 5' antisense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second double-stranded inhibitory RNA molecule, xi) The 5' antisense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, xiiii) The 3' antisense strand of the first double-stranded inhibitory RNA and the 5' sense strand of the second double-stranded inhibitory RNA molecule, xiv) The 3' antisense strand of the first double-stranded inhibitory RNA and the 3' sense strand of the second double-stranded inhibitory RNA molecule, xv) The 3' antisense strand of the first double-stranded inhibitory RNA and the 3' antisense strand of the second double-stranded inhibitory RNA molecule, xvi) A nucleic acid molecule selected from the group consisting of the 3' antisense strand of the first double-stranded inhibitory RNA and the 5' antisense strand of the second double-stranded inhibitory RNA molecule.

3. The nucleic acid molecule according to claim 1 or 2, wherein the first and second genes to be silenced are different genes.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is the ApoB gene.

5. The nucleic acid molecule according to claim 4, wherein the Apo B gene contains the nucleotide sequence described in Sequence ID No. 1, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

6. A nucleic acid molecule according to claim 5, wherein the Apo B double-strand inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 607 to 906.

7. A nucleic acid molecule according to claim 5, wherein the Apo B double-stranded inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 907 to 1206.

8. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is DGAT2.

9. The nucleic acid molecule according to claim 8, wherein the DGAT2 gene contains the nucleotide sequence described in SEQ ID NO: 6, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

10. A nucleic acid molecule according to claim 9, wherein the double-stranded DGAT2 inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 to 306.

11. A nucleic acid molecule according to claim 9, wherein the double-stranded DGAT2 inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 307 to 606.

12. The nucleic acid molecule according to claim 9, wherein the double-stranded DGAT2 inhibitory RNA comprises or consists of the antisense nucleotide sequence described in Sequence ID No. 4830.

13. The nucleic acid molecule according to claim 9, wherein the double-stranded DGAT2 inhibitory RNA contains or consists of the sense nucleotide sequence described in Sequence ID No. 4829.

14. A nucleic acid molecule according to claim 9, wherein the double-stranded DGAT2 inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4840 to 4848.

15. A nucleic acid molecule according to claim 9, wherein the double-stranded DGAT2 inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4831 to 4839.

16. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is PCSK9.

17. The nucleic acid molecule according to claim 16, wherein the PCSK9 gene contains the nucleotide sequence described in Sequence ID No. 2, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

18. A nucleic acid molecule according to claim 17, wherein the double-stranded PCSK9 inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 1207 to 1510.

19. A nucleic acid molecule according to claim 17, wherein the double-stranded PCSK9 inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 1511 to 1814.

20. The nucleic acid molecule according to claim 17, wherein the double-stranded PCSK9 inhibitory RNA comprises or consists of the antisense nucleotide sequence described in Sequence ID No. 4822.

21. The nucleic acid molecule according to claim 17, wherein the double-stranded PCSK9 inhibitory RNA contains or consists of the sense nucleotide sequence shown in Sequence ID No. 4821.

22. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is lipoprotein A.

23. The nucleic acid molecule according to claim 22, wherein the lipoprotein A gene contains the nucleotide sequence described in Sequence ID No. 3, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

24. A nucleic acid molecule according to claim 23, wherein the lipoprotein A double-strand inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 1815 to 2114.

25. A nucleic acid molecule according to claim 23, wherein the lipoprotein A double-strand inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 2115 to 2414.

26. The nucleic acid molecule according to claim 23, wherein the lipoprotein A double-strand inhibitory RNA contains or consists of the antisense nucleotide sequence described in Sequence ID No. 4824.

27. The nucleic acid molecule according to claim 23, wherein the lipoprotein A double-strand inhibitory RNA contains or consists of the sense nucleotide sequence described in Sequence ID No. 4823.

28. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene is angiotensinogen.

29. The nucleic acid molecule according to claim 28, wherein the angiotensinogen gene contains the nucleotide sequence described in Sequence ID No. 4, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

30. A nucleic acid molecule according to claim 29, wherein the double-stranded angiotensinogen inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 2415 to 2714.

31. A nucleic acid molecule according to claim 29, wherein the double-stranded angiotensinogen inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 2715 to 3014.

32. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is Apo CIII.

33. The nucleic acid molecule according to claim 32, wherein the Apo CIII gene contains the nucleotide sequence described in Sequence ID No. 5, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

34. A nucleic acid molecule according to claim 33, wherein the double-stranded Apo CIII inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3015 to 3314.

35. A nucleic acid molecule according to claim 33, wherein the double-stranded Apo CIII inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3315 to 3614.

36. The nucleic acid molecule according to claim 33, wherein the double-stranded ApoCIII inhibitory RNA comprises or consists of the sense nucleotide sequence described in Sequence ID No. 4860.

37. The nucleic acid molecule according to claim 33, wherein the double-stranded ApoCIII inhibitory RNA contains or consists of the antisense nucleotide sequence described in Sequence ID No. 4859.

38. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is ANGPTL3.

39. The nucleic acid molecule according to claim 38, wherein the ANGPTL3 gene comprises the nucleotide sequence shown in Sequence ID No. 4819, and the double-stranded inhibitory RNA is 19 to 23 nucleotides long.

40. A nucleic acid molecule according to claim 39, wherein the double-stranded ANGPTL3 inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3615 to 3914.

41. A nucleic acid molecule according to claim 39, wherein the double-stranded ANGPTL3 inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 3915 to 4214.

42. A nucleic acid molecule according to claim 39, wherein the double-stranded ANGPTL3 inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NO: 4826 or 4828.

43. A nucleic acid molecule according to claim 39, wherein the double-stranded ANGPTL3 inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NO: 4825 or 4827.

44. The nucleic acid molecule according to any one of claims 1 to 3, wherein the gene to be silenced is ANGPTL4.

45. The nucleic acid molecule according to claim 44, wherein the ANGPTL4 gene comprises the nucleotide sequence shown in Sequence ID No. 4820, and the double-stranded inhibitory RNA has a length of 19 to 23 nucleotides.

46. A nucleic acid molecule according to claim 45, wherein the ANGPTL4 double-stranded inhibitory RNA contains or comprises a sense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4215 to 4514.

47. A nucleic acid molecule according to claim 45, wherein the ANGPTL4 double-stranded inhibitory RNA contains or comprises an antisense nucleotide sequence selected from the group consisting of SEQ ID NOs: 4515 to 4814.

48. A nucleic acid molecule according to any one of claims 1 to 35, wherein the first or second gene to be silenced is selected from the group consisting of Apo B, DGAT2, PCSK9, Lp(a), APOCIII, angiotensinogen, ANGPTL3, and ANGPTL4.

49. The nucleic acid molecule according to claim 48, wherein the first gene is Apo B and the second gene is DGAT2.

50. The nucleic acid molecule according to any one of claims 1 to 49, wherein the single-stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815).

51. The nucleic acid molecule according to any one of claims 1 to 50, wherein the complementary single-stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816).

52. The nucleic acid molecule according to claim 50, wherein the single-stranded DNA contains the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is bound to the 5' end of the sense nucleotide sequence.

53. The nucleic acid molecule according to claim 50, wherein the single-stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is bound to the 5' end of the antisense nucleotide sequence.

54. The nucleic acid molecule according to claim 51, wherein the complementary single-stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is bound to the 5' end of the sense nucleotide sequence.

55. The nucleic acid molecule according to claim 50, wherein the complementary single-stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is bound to the 5' end of the antisense nucleotide sequence.

56. The nucleic acid molecule according to any one of claims 1 to 55, wherein the first and / or second double-strand inhibitory (RNA) molecule comprises a modified nucleotide and / or a modified sugar(s).

57. The nucleic acid molecule according to any one of claims 1 to 56, wherein the first and / or second double-strand inhibitory (RNA) molecule comprises at least one modified nucleotide, the modification being 2'-deoxy-2'-fluoro.

58. The nucleic acid molecule according to any one of claims 1 to 57, wherein the first and / or second double-stranded inhibitory RNA molecule comprises at least one modified nucleotide, the modification being 2'-O-methyl.

59. The nucleic acid molecule according to any one of claims 1 to 58, wherein the first and / or second double-strand inhibitory (RNA) molecule comprises at least one modified nucleotide and the modified 5'-vinyl phosphate.

60. The nucleic acid molecule according to any one of claims 1 to 59, wherein the first and / or second double-stranded inhibitory RNA molecule comprises at least one phosphorothioate bond.

61. The nucleic acid molecule according to any one of claims 1 to 60, wherein the first and / or second double-strand inhibitory (RNA) molecule comprises at least one modified sugar.

62. The nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 61, wherein the nucleic acid molecule comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830), and the single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

63. The nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 61, wherein the nucleic acid molecule comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand (SEQ ID NO: 4821) and an antisense strand (SEQ ID NO: 4822), and the single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

64. The nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 61, wherein the nucleic acid molecule comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand (SEQ ID NO: 4823) and an antisense strand (SEQ ID NO: 4824), and the single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

65. The nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 61, wherein the nucleic acid molecule comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826), and the single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

66. The nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 61, wherein the nucleic acid molecule comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand (SEQ ID NO: 4827) and an antisense strand (SEQ ID NO: 4828), and the single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

67. The nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 61, wherein the nucleic acid molecule comprises at least one double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand (SEQ ID NO: 4860) and an antisense strand (SEQ ID NO: 4859), and the single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' sense strand.

68. A nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 67, wherein the nucleic acid molecule is i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4821) and an antisense strand (SEQ ID NO: 4822), wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A nucleic acid molecule comprising: a second nucleic acid comprising a double-stranded inhibitory (RNA) molecule having a sense strand (SEQ ID NO: 4823) and an antisense strand (SEQ ID NO: 4824), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-stranded inhibitory (RNA) molecules.

69. A nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 67, wherein the nucleic acid molecule is i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826), wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A nucleic acid molecule comprising a second nucleic acid, which includes a double-stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-stranded inhibitory (RNA) molecules.

70. A nucleic acid molecule according to any one of claims 1 to 52, 54, and 56 to 67, wherein the nucleic acid molecule is i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule including a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830), wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A nucleic acid molecule comprising a second nucleic acid, which includes a double-stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4860) and an antisense strand (SEQ ID NO: 4859), wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, and the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-stranded inhibitory (RNA) molecules.

71. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4849, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4822.

72. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4850, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4822.

73. A nucleic acid molecule according to any one of claims 1 to 61, wherein the acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4851, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4824.

74. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4852, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4824.

75. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4853, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4826.

76. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4854, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4826.

77. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4855, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4828.

78. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4856, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4828.

79. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4857, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4830.

80. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4858, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4830.

81. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4861, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4859.

82. The nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid comprises a sense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4862, and an antisense strand containing or consisting of the nucleotide sequence shown in SEQ ID NO: 4859.

83. A nucleic acid molecule according to any one of claims 1 to 61, wherein the nucleic acid molecule is i) A first nucleic acid comprising a double-stranded inhibitory ribonucleic acid (RNA) molecule having a sense strand selected from the group consisting of SEQ ID NOs. 607 to 906 and an antisense strand selected from the group consisting of SEQ ID NOs. 907 to 1206, wherein a single-stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5' sense strand is positioned on the first nucleic acid, ii) A nucleic acid molecule comprising: a second nucleic acid comprising an antisense strand selected from the group consisting of SEQ ID NOs. 7-306, 4829 and 4831-4839, and an antisense strand selected from the group consisting of SEQ ID NOs. 307-606, 4830 and 4840-4848, wherein a single-stranded deoxyribonucleic acid (DNA) molecule is conjugated to the 5' of the sense strand, the single-stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single-stranded deoxyribonucleic acid (DNA) molecule described in i) above, and is annealed by complementary base pairing to form a double-stranded DNA linker that links the first and second double-stranded inhibitory ribonucleic acid (RNA) molecules.

84. The nucleic acid molecule according to claim 83, wherein the second nucleic acid comprises a double-strand inhibitory RN molecule comprising a sense strand shown in SEQ ID NO: 4857 and an antisense strand shown in SEQ ID NO: 4830.

85. The nucleic acid molecule according to claim 83, wherein the second nucleic acid comprises a double-strand inhibitory (RNA) molecule comprising a sense strand shown in SEQ ID NO: 4858 and an antisense strand shown in SEQ ID NO: 4830.

86. The nucleic acid molecule according to any one of claims 1 to 85, wherein the nucleic acid molecule is covalently bonded to N-acetylgalactosamine.

87. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 86, and comprising a pharmaceutical carrier and / or an excipient.

88. A nucleic acid molecule or pharmaceutical composition according to any one of claims 1 to 87, for use in the treatment or prevention of a subject having hypercholesterolemia or a predisposition to hypercholesterolemia.

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