Dual silencing

Abstract

A nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference t

Description

24 Field of the Disclosure 5 The disclosure relates to isolated nucleic acid molecules comprising at least two double stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence by RNA interference angiopoietin like 3 (ANGPLT3) and diacylglycerol O acyltransferase 2 (DGAT2) to enhance silencing thereby modulating gene expression. 10 Background to the Disclosure A technique to specifically ablate gene function is through the introduction of double stranded inhibitory RNA, also referred to as small inhibitory or interfering RNA (siRNA), 15 into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA molecule. The siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The siRNA molecule is typically, but not exclusively, derived from exons of the gene which is to be ablated. Many organisms respond to the 20 presence of double stranded RNA by activating a cascade that leads to the formation of siRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA 25 complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA. Typically, RNA silencing utilises a single siRNA species directed to a single gene to silence expression. It is known to link siRNAs to enable the silencing of two or more 30 genes. For example, WO2016205410 discloses oligonucleotides linked together directly, via functional end-substitutions, or indirectly by way of a linking agent. The oligonucleotide can be bound directly to a linker. Such bonding can be achieved, for example, through use of 3'-thionucleosides. WO2018145086 discloses oligonucleotides in the form of a multimeric oligonucleotide having monomeric subunits of oligonucleotide 35 joined by covalent linkers to decrease clearance due to glomerular filtration. WO2013040429A1 discloses multi-oligomeric complexes that comprise two or more targeting oligonucleotides linked together by a cleavable linker. WO2015113922 24 discloses oligo oligonucleotides conjugates where two or more antisense oligonucleotides are covalently linked by physiologically labile linkers, and to a biocleavable functional group such as a conjugate group. WO2010141511 discloses bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules 5 having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and / or in which the target regions are complementary to more than one target gene or target sequence. WO2017188707 discloses a dicer substrate RNA nanostructure exhibiting enhanced gene silencing effects. The RNA nanostructure includes a plurality of the same 10 or different RNAi sequences in a single RNA nanostructure. The use of oligomeric nanostructures comprising more than one siRNA or antisense molecule is known in the art. There is a desire to provide alternative and simpler approaches to the delivery of more 15 than one inhibitory RNA to a cell with the objective of silencing the expression of one or more gene targets to obtain enhanced therapeutic effects. Cardiovascular disease associated with hypercholesterolemia is a common condition and results in heart disease and a high incidence of death and morbidity and can be a 20 consequence of poor diet, obesity, or an inherited dysfunctional gene. For example, mutations in Low Density Lipoprotein Receptor (LDL-receptor) or apolipoprotein B (ApoB) as in familial hypercholesterolemia. Cholesterol is essential for membrane biogenesis in animal cells. The lack of water solubility means that cholesterol is transported around the body in association with lipoproteins. Apolipoproteins form 25 together with phospholipids, cholesterol and lipids which facilitate the transport of lipids such as cholesterol, through the bloodstream to the different parts of the body. Lipoproteins are classified according to size and can form HDL (High-density lipoprotein), LDL (Low-density lipoprotein), IDL (intermediate-density lipoprotein), VLDL (very low-density lipoprotein) and ULDL (ultra-low-density lipoprotein) lipoproteins. 30 Familial hypercholesterolemia is an orphan disease and results from elevated levels of LDL cholesterol (LDL-C) in the blood. The disease is an autosomal dominant disorder with both the heterozygous (350-550mg / dL LDL-C) and homozygous (650-1 OOOmg / dL LDL-C) states resulting in elevated LDL-C. The heterozygous form of familial 35 hypercholesterolemia is around 1:500 of the population. The homozygous state is much rarer and is approximately 1:1,000,000. The normal levels of LDL-C are in the region 130mg / dL. 24 Hypercholesterolemia is particularly acute in paediatric patients which if not diagnosed early can result in accelerated coronary heart disease and premature death. If diagnosed and treated early the child can have a normal life expectancy. In adults, high 5 LDL-C, either because of mutation or other factors, is directly associated with increased risk of atherosclerosis which can lead to coronary artery disease, stroke or kidney problems. Lowering levels of LDL-C is known to reduce the risk of atherosclerosis and associated conditions. LDL-C levels can be lowered initially by administration of statins which block the de novo synthesis of cholesterol by inhibiting the HMG-CoA reductase. 10 Some subjects can benefit from combination therapy which combines a statin with other therapeutic agents such as ezetimibe, colestipol or nicotinic acid. However, expression and synthesis of HMG-CoA reductase adapts in response to the statin inhibition and increases over time, thus the beneficial effects are only temporary or limited after statin resistance is established. 15 The disclosure relates to isolated nucleic acid molecules comprising at least two double stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence by RNA interference either the same gene or different genes to enhance silencing thereby modulating gene expression. 20 Statements of Invention According to an aspect of the invention there is provided a nucleic acid molecule comprising: 25 i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a cardiovascular disease associated gene to be silenced wherein said cardiovascular gene is diacylglycerol O acyltransferase 2 (DGAT 30 2) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a different cardiovascular disease associated gene to be silenced wherein said different 35 cardiovascular gene is angiopoietin like 3 (ANGPLT 3) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is 24 substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules. 5 In a preferred embodiment of the invention said DGAT2 gene comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. 10 Ina preferred embodiment of the invention said double stranded DGAT2 inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 7 to 306. In a preferred embodiment of the invention said double stranded DGAT2 inhibitory RNA 15 comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 307 to 606. In a preferred embodiment of the invention said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 20 4830. In a preferred embodiment of the invention said double stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence set forth in SEQ ID NO: 4829. 25 In a preferred embodiment of the invention said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4840 to 4848. In a preferred embodiment of the invention said double stranded DGAT2 inhibitory RNA 30 comprises or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 4831 to 4839. In a preferred embodiment of the invention said ANGPTL3 gene comprises a nucleotide sequence set forth in SEQ ID NO: 4819 wherein said double stranded inhibitory RNA is 35 19-23 nucleotides in length. 24 In a preferred embodiment of the invention said double stranded ANGPTL 3 inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 3615 to 3914. 5 In a preferred embodiment of the invention said double stranded ANGPTL 3 inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 3915 to 4214. In a preferred embodiment of the invention said double stranded ANGPTL3 inhibitory 10 RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4826 or 4828. In a preferred embodiment of the invention said double stranded ANGPTL3 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group: 15 SEQ ID NO: 4825 or 4827. In an alternative preferred embodiment of the invention said first gene is DGAT2 and comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length and wherein said double stranded 20 DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4830 or 4840 to 4848, and / or a sense nucleotide sequence set forth in SEQ ID NO: 4829 or 4831 to 4839. In a preferred embodiment of the invention said single stranded DNA comprises the 25 nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815). Preferably said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816). 30 In a preferred embodiment of the invention said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the sense nucleotide sequence. In an alternative preferred embodiment of the invention said single stranded DNA 35 comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the antisense nucleotide sequence. 24 In a preferred embodiment of the invention said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the sense nucleotide sequence. 5 In a preferred embodiment of the invention said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the antisense nucleotide sequence. In a preferred embodiment of the invention said first and / or said second double stranded 10 inhibitory RNA molecule comprises or consists of natural nucleotides. In an alternative preferred embodiment of the invention said first and / or said second double stranded inhibitory RNA molecule comprises modified nucleotides and / or modified sugar(s). 15 In a preferred embodiment of the invention said modified nucleotides / sugars are selected from the group: a 3 '-terminal deoxy- thymine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a 20 constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2' -hydroxyl- modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0- alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a 25 cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising phosphorodithioate (PS2), a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5’- phosphate, and a nucleotide comprising a 5 ‘-phosphate mimic, for example a 5’-vinyl phosphate, a nucleotide comprising a 2’-deoxy-2’-fluro and a 2’ methyl sugar base and glycol nucleic acid. 30 In a preferred embodiment of the invention said first or and / or second double stranded inhibitory RNA molecule comprises at least one modified nucleotide wherein said modification is 2'-deoxy-2'-fluoro. 35 In a preferred embodiment of the invention said first and / or said second double stranded inhibitory RNA molecule comprises at least one modified nucleotide wherein said modification is 2'-O-methyl. 24 In a further preferred embodiment of the invention said first and / or said second double stranded inhibitory RNA molecule comprises at least one phosphorothioate linkage. 5 Ina further preferred embodiment of the invention said first and / or said second double stranded inhibitory RNA molecule comprises at least one 5'-vinyl phosphate. In an embodiment of the invention said first and / or said second double stranded inhibitory RNA molecule comprises at least one modified sugar. 10 A sugar modification includes a modified version of the ribosyl moiety, such as -O-modified RNA such as 2'-0-alkyl or 2'-0-(substituted)alkyl e.g. 2'-0-methyl, T-0-(2-cyanoethyl), 2'-0-(2-methoxy)ethyl (2-MOE), 2'-0-(2-thiomethyl)ethyl, 2'-0-butyryl, -0-propargyl, 2'-0-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-15 amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkoxy)methyl, e.g. 2'-0-(2-chloroethoxy)methyl (MCEM), -0- (2,2-dichloroethoxy)methyl (DCEM); 2'-<3-alkoxycarbonyl e.g. T-0-[2- (methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), T-0-[2-(N,N- dimethylcarbamoyl)ethyl] (DOME); 2'-halo e.g. 2'-F, FANA (2'-F arabinosyl nucleic acid); carbasugar and azasugar modifications; 3 20 '-O-alkyl e.g. 3'-0-methyl, 3 '-O-butyryl, V-0- propargyl and their derivatives. In a preferred embodiment of the invention there is provided a nucleic acid molecule according to the invention wherein said nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand 25 (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand. In a preferred embodiment of the invention there is provided a nucleic acid molecule according to the invention wherein said nucleic acid molecule comprises at least one 30 double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand. In a preferred embodiment of the invention there is provided a nucleic acid molecule 35 according to the invention wherein said nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand 24 (SEQ ID NO: 4827) and an antisense strand (SEQ ID NO: 4828) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand. In a preferred embodiment of the invention there is provided a nucleic acid molecule 5 comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826) wherein there is provided a single stranded deoxyribonucleic acid (DNA) 10 molecule conjugated to the 5’ sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ of said sense strand wherein the single stranded 15 deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules. 20 In a preferred embodiment of the invention said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815). Preferably said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816). 25 In a preferred embodiment of the invention said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the sense nucleotide sequence. 30 In an alternative preferred embodiment of the invention said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the antisense nucleotide sequence. In a preferred embodiment of the invention said single stranded DNA comprises the 35 nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the sense nucleotide sequence. 24 In a preferred embodiment of the invention said single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the antisense nucleotide sequence. 5 In an alternative further preferred embodiment of the invention said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4854 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4826. 10 Ina further preferred embodiment of the invention said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4855 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4828. 15 In an alternative further preferred embodiment of the invention said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4856 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4828. 20 In an alternative further preferred embodiment of the invention said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4857 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4830. 25 In an alternative further preferred embodiment of the invention said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4858 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4830 30 In a preferred embodiment of the invention said second nucleic acid comprises a double stranded inhibitory (RNA) molecule comprising a sense strand set forth in SEQ ID NO 4857 and an antisense strand set forth in SEQ ID NO 4830. In an alternative preferred embodiment of the invention said second nucleic acid 35 comprises a double stranded inhibitory (RNA) molecule comprising a sense strand set forth in SEQ ID NO 4858 and an antisense strand set forth in SEQ ID NO 4830. 24 In a preferred embodiment of the invention said nucleic acid molecule is covalently linked to A / -acetylgalactosamine. The sugar moiety in N-acetylgalactosamine can comprise glycosidic linkages to improve 5 stability. A variety of glycosidic bonds are known in the art and formed between the hemiacetal of the sugar moiety and several chemical groups forming O-, N-, S- or C-glycosidic bonds. Preferably the N-acetylgalactosamine comprises an 0-, N-, S- or C-glycosidic bond. 10 In a further embodiment of the invention said N-acetylgalactosamine is linked to either the antisense part of said inhibitory RNA or the sense part of said inhibitory RNA. In a further embodiment of the invention said N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA 15 molecule of said first nucleic acid. In a further embodiment of the invention said N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said second nucleic. 20 In a further embodiment of the invention N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said first nucleic acid and is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said second nucleic. 25 Preferably, N-acetylgalactosamine is linked to the 3’ terminus is of said sense RNA. In an alternative embodiment of the invention N-acetylgalactosamine is linked to the 5’ terminus of said sense RNA. 30 In an alternative preferred embodiment of the invention said N-acetylgalactosamine is linked to the 3’ terminus of said antisense RNA. In a preferred embodiment of the invention N-acetylgalactosamine is monovalent. 35 In a preferred embodiment of the invention N-acetylgalactosamine is divalent. In an alternative embodiment of the invention N-acetylgalactosamine is trivalent. In a preferred embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: AcHN In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: In an alternative embodiment of the invention said nucleic acid molecule is covalently 15 linked to a molecule comprising the structure: In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: 5 In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: 10 AcHN In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: 15 According to a further aspect of the invention there is provided a medicament comprising a nucleic acid according to the invention. 24 5 According to a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid molecule according to the invention. In a preferred embodiment of the invention said composition further includes a pharmaceutical carrier and / or excipient. 10 According to a further aspect of the invention there is provided a nucleic acid molecule or a pharmaceutical composition according to the invention for use in the treatment or prevention of a subject that has or is predisposed to hypercholesterolemia. 15 In a preferred embodiment of the invention said use is the treatment or prevention of diseases associated with hypercholesterolemia selected from the group stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, aortic stenosis, cerebrovascular disease, peripheral arterial disease, 20 hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Buerger’s disease, renal artery stenosis, hyper-apobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis. 25 When administered the compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and optionally other therapeutic agents, such as cholesterol lowering agents, which can be administered separately from the nucleic acid molecule according 30 to the invention or in a combined preparation if a combination is compatible. The combination of a nucleic acid according to the invention and the other, different therapeutic agent is administered as simultaneous, sequential, or temporally separate dosages. 35 The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, 24 be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal ortransepithelial. The compositions of the invention are administered in effective amounts. An “effective 5 amount” is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of treating a disease, such as cardiovascular disease, the desired response is inhibiting or reversing the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. 10 This can be monitored by routine methods. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy 15 (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It 20 will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. The pharmaceutical compositions used in the foregoing methods preferably are sterile 25 and contain an effective amount of a nucleic acid molecule according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by determining regression of cardiovascular disease and decrease of disease symptoms etc. 30 The doses of the nucleic acid molecule according to the invention administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. If a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more 35 localized delivery route) may be employed to the extent that patient tolerance permits. It will be apparent that the method of detection of the nucleic acid according to the 24 invention facilitates the determination of an appropriate dosage for a subject in need of treatment. In general, doses of the nucleic acid molecules herein disclosed of between 1nM - 1pM 5 generally will be formulated and administered according to standard procedures. Preferably doses can range from 1nM- 500nM, 5nM-200nM, 10nM-100nM. Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. The administration of compositions 10 to mammals other than humans, (e.g., for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a nonhuman primate, cow, horse, pig, sheep, goat, dog, cat or rodent. 15 When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible 20 carriers, and optionally other therapeutic agents e.g. statins. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the 25 following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. 30 Compositions may be combined, if desired, with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term “pharmaceutically acceptable carrier” in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the 35 active ingredient is combined to facilitate, for example, solubility and / or stability. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that 24 there is no interaction which would substantially impair the desired pharmaceutical efficacy. The pharmaceutical compositions may contain suitable buffering agents, including acetic 5 acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All 10 methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. 15 Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of nucleic acid, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile 20 injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butane diol. Among the acceptable solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may 25 be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. 30 Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the 35 function of the essential integers. 24 Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. 5 Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. 10 An embodiment of the invention will now be described by example only and with reference to the following materials, methods and examples: MATERIALSAND METHODS 15 Transfection in primary mouse hepatocytes Duplex siRNAs and heterodimer siRNAs synthesized by Bio-Synthesis (Lewisville, TX) (Table 3 and 5), were resuspended in nuclease-free water (Invitrogen™ AM9932) to generate a stock solution of 10 pM. For serum stability assay, stock siRNAs were 20 incubated in vehicle (nuclease-free water) or in various concentrations (20% - 80%) of human serum (HS) for 2 hours at 37°C. After pre-incubation in serum or vehicle, siRNAs were transfected into primary mouse hepatocytes in triplicates in a 384-well plate (Thermo Scientific™ 164688) at various concentrations ranging from 2.5 nM to 100 nM using 0.15 pL of Lipofectamine RNAiMAX (Invitrogen™ 13778075) per well. Transfected 25 cells were incubated at 37°C and 5% CO2 for 48 hours. Cells receiving no siRNA treatment were used as control. Free-uptake assay of GalNAc-conjugated ApoB-mTTR heterodimers in primary mouse hepatocytes 30 Standard siRNA controls (mTTR and ApoB_C3_01) and GalNAc-conjugated ApoB-mTTR heterodimers (Table 7) were synthesized at Bio-Synthesis (Lewisville, TX). GalNAc-conjugated constructs and standard siRNA controls were resuspended in nuclease-free water (Invitrogen™ AM9932) to generate a stock solution of 10 pM. Stock 35 siRNAs were distributed in triplicates in 384-well plates (Thermo Scientific™ 164688) to a final concentration of 4 nM, 25 nM and 100 nM and primary mouse hepatocytes were added at a concentration of 5,000 cells per well for free-uptake assay. After treatment, 24 cells were incubated at 37°C and 5% CO2 for 48 hours. Cells receiving no siRNA treatment were used as control. Duplex RT-qPCR 5 Cells were processed for RT-qPCR read-out using the Cells-to-CT 1-step TaqMan Kit (Invitrogen™ A25603). Briefly, cells were washed with 50pL ice-cold PBS and lysed in 20 pl Lysis solution containing DNase I. Lysis was stopped after 5 minutes by addition of 2 pl STOP Solution for 2 min. For the RT-qPCR analysis, 1 pL of lysate was dispensed per well into a 96-well PCR plate in a 10 pL RT-qPCR reaction volume. RT-qPCR was 10 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 15 was determined using the AACT method, where GAPDH was used as internal control and expression changes normalized to the reference sample (no siRNA treatment). Receptor-mediated uptake assays of GalNAc-conjugated ApoB-DGAT2 heterodimers vs single siRNA counterparts 20 GalNAc-conjugated ApoB (CS13) and DAGT2 (CS8) single siRNA controls and ApoB-DGAT2 (CS15 &CS16) heterodimer siRNAs (Table 3), were synthesized at CatSci (Cardiff, UK). Constructs were resuspended in nuclease-free water (Invitrogen™ AM9932) to generate a stock solution of 100 pM. 25 Freshly prepared or *serum pre-incubated stock siRNAs were distributed in triplicates in collagen-coated 384-well plates (Corning™ 354666) to a final concentration of 0.1 nM, 1 nM, 4 nM and 25 nM and primary mouse hepatocytes were added at a concentration of 6,000 cells per well. After treatment, cells were incubated at 37°C and 5 % CO2 for 48 30 hours. Cells receiving no siRNA treatment were used as control. (*Stock siRNAs were exposed to 80 % human serum (HS) for 2 hours at 37°C). Duplex RT-qPCR 35 Cells were processed for RT-qPCR read-out using the Cells-to-CT 1-step TaqMan Kit (Invitrogen™ A25603). Briefly, cells were washed with 50 pL ice-cold PBS and lysed in 20 pl Lysis solution containing DNase I. Lysis was stopped after 5 minutes by addition of 24 2 pl STOP Solution for 2 min. For the RT-qPCR analysis, 1 pL of lysate was dispensed per well into a 96-well PCR plate in a total of 10 pL 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), 5 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 AACT method, where GAPDH was used as internal control and expression changes normalized to the reference sample (no siRNA treatment). 10 In vivo mouse study Methodology Male C57BL / 6J mice (20-25 g) were group housed in the Saretius animal unit at the 15 University of Reading, and maintained under a 12 h light / dark cycle, at 23°C with humidity controlled according to Home Office regulations. Mice were given access to standard rodent chow SDS rat expanded diet (RM3-E-FG) for the duration of the study. Formulation of siRNA compounds 20 Heterodimer compounds CS15 and CS16 (Table 3) were each formulated in RNase free PBS to concentrations of 2 and 5 mg / mL to provide doses of 10 and 20 mg / kg when given subcutaneously (SC) in a 5 mL / kg dosing volume. Single siRNA control compounds CS8, CS13 and PAR-ApoB control (Table 3) were each formulated in 25 RNAase free PBS to concentrations of 1 and 2 mg / mL to provide doses of 5 and 10 mg / kg when given SC in 5 mL / kg dosing volumes. 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 given SC in 5 mL / kg dosing volume. 30 Liver processing for RT-qPCR At Day 5 and Day 14 following siRNA compound or Vehicle injection (n=4), each treatment group was terminally sampled by cardiac puncture under isoflurane. Liver tissue was excised and snap frozen in liquid N2. Total RNA was extracted from 35 homogenates of snap-frozen whole liver using QIAGEN RNeasy Mini Kit (74104). 24 Duplex RT-qPCR was performed using the ThermoFisher TaqMan Fast 1-Step Master Mix with TaqMan probes forGAPDH (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 5 determined using the AACT method, where GAPDH was used as internal control and the expression changes of the target gene were normalized to the vehicle control. SEQ ID correspondence: SEQ ID NO Description 1 ApoB 2 PCSK9 3 Lp(a) 4 Angiotensinogen 5 APOCIII 6 DGAT2 7-306 DGAT2 sense 307-606 DGAT2 antisense 607-906 ApoB sense 907-1206 ApoB antisense 1207-1510 PCSK9 sense 1511-1814 PCSK9 antisense 1815-2114 Lp(a) sense 2115-2414 Lp(a) antisense 2415-2714 Angiotensinogen sense 2715-3014 Angiotensinogen antisense 3015-3314 APOCIII sense 3315-3614 APOCIII antisense 3615-3914 ANGPTL3 sense 3915-4214 ANGPTL3 antisense 4215-4514 ANGPTL4 sense 4515-4814 ANGPTL4 antisense 4815 Single stranded DNA 4816 Single stranded DNA 4817 Single stranded DNA 4818 Single stranded DNA 24 4819 ANGPTL3 4820 ANGPTL4 4821 PCSK9 sense 4822 PCSK9 antisense 4823 Lp(a) sense 4824 Lp(a) antisense 4825 ANGPTL3 sense 4826 ANGPTL3 antisense 4827 ANGPTL3 sense 4828 ANGPTL3 antisense 4829 DGAT2 sense 4830 DGAT2 antisense 4831-4839 DGAT2 sense 4840-4848 DGAT2 antisense 4849-4850 PCSK9 sense crook and anticrook 4851-4852 Lp(a) sense crook and anticrook 4853-4854 ANGPTL3 sense crook and anticrook 4855-4856 ANGPTL3 sense crook and anticrook 4857-4858 DGAT 2 sense crook and anticrook 4859 APOCI 11 antisense 4860 APOCIII sense 4861-4862 APOCI 11 crook and anticrook Example 1 5 As shown in Table 1, when transfected in primary mouse hepatocytes at concentrations of 2.5-100 nM to test efficacy, ApoB_C3_S19 and ApoB_C3_S18 (single siRNAs with hybridized complementary DNA at the 5' end of the sense strand - Table 3) showed knockdown of ApoB comparable to ApoB_C3_OR with no complementary DNA 10 hybridized to the DNA crook at the 5’ end of the sense strand. As for the ApoB-mTTR heterodimer, the mTTR portion showed knockdown of mTTR target comparable to the modified single siRNA mTTR (bottom 2 rows in Table 1) while the ApoB portion showed reduced efficacy (KD range: 64.2% - 76.3%) compared to the single siRNA counterpart ApoB_C3_OR (KD range: 90.1% - 92.8%). 15 To test stability in human serum, all the siRNA constructs were preincubated in human serum for 2 hours and transfected in primary mouse hepatocytes for 48 hours when knockdown of target gene was measured by RT-qPCR. As shown in Table 2, the mTTR portion in the ApoB-mTTR heterodimer was stable across different concentrations of human serum showing KD levels between 87% and 94.5% which were comparable to KD levels of the standard mTTR siRNA (KD between 74.3% and 93.1%). As for the ApoB portion of the ApoB-mTTR heterodimer siRNA, serum stability was high in 20% human serum showing 92.1% and 84.7% KD at 2.5 nM and 25 nM respectively, while KD was reduced after incubation in 80% human serum showing values of 32.5% and 23.9% KD at 2.5 nM and 25 nM respectively. In contrast, the standard ApoB siRNA counterpart ApoB_C3_OR showed stable KD ranging from 82.4% to 97.6% across different serum concentrations (Table 2). 24 Table 1. Efficacy of ApoB-mTTR heterodimer siRNA and standard siRNAs siRNA ID KD levels (%) in no serum preincubation 2.5 nM 10 nM 25 nM 100 nM ApoB_C3_OR 90.1 90.2 90.7 92.8 ApoB_C3_S19 89.0 88.4 88.5 90.6 ApoB C3 S18 84.6 90.6 91.6 92.0 ApoB-mTTR (ApoB) 76.3 71.5 71.1 64.2 ApoB-mTTR (mTTR) 92.4 94.3 95.3 96.2 mTTR 92.3 94.1 95.9 95.3 Table 2. Serum stability of ApoB-mTTR heterodimer siRNA and standard siRNAs siRNA ID KD levels (%) after preincubation in human serum 2.5 nM + 20% HS 25 nM + 20% HS 2.5 nM + 50% HS 25 nM + 50% HS 2.5 nM + 80% HS 25 nM + 80% HS ApoB_C3_OR 97.6 96.8 94.4 95.5 82.4 91.3 ApoB_C3_S19 96.8 95.4 86.5 89.0 59.3 84.7 ApoB_C3_S18 96.6 95.2 90.4 90.4 74.1 83.6 ApoB-mTTR (ApoB) 92.1 84.7 80.0 66.8 32.5 23.9 ApoB-mTTR (mTTR). 87.2 87.4 89.2 87.0 93.9 94.5 mTTR 82.9 89.3 74.3 84.0 89.2 93.1 Table 3. siRNA and ApoB-mTTR heterodimer siRNA description siRNA ID Sense (5' - 3') Antisense (5' - 3') 24 ApoB_C3_OR CGAAGCGCCCTACTCCACT G*G*UGU AUG GCU UCA ACC CUG A U*C*A GGG UUG AAG CCA UAC ACC*U*U ApoB_C3_S1 9 CGAAGCGCCCTACTCCACT G*G*UGU AUG GCU UCA ACC CUG A 1 U*C*A GGG UUG AAG CCA UAC ACC*U*U ApoB_C3_S1 8 CGAAGCGCCCTACTCCACT G*G*UGU AUG GCU UCA ACC CUG A 2 U*C*A GGG UUG AAG CCA UAC ACC*U*U ApoB-mTTR heterodimer ApoB CGAAGCGCCCTACTCCACT G*G*UGU AUG GCU UCA ACC CUG A 3 U*C*A GGG UUG AAG CCA UACACC*U*U mTTR AGTGGAGTAGGGCGCTTCG fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA 3 mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU mTTR fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA - GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU 1 AGTGGAGTAGGGCGCTTCG (SEQ ID NO 4816; 19 nt DNA fragment hybridised with complementary DNA at the 5' end of ApoB_C3_S19) 2 GTGGAGTAGGGCGCTTCG (SEQ ID NO 4817) 18 nt DNA fragment hybridised with complementary DNA at the 5' end of ApoB_C3_S18) 3 DNA sequence CGAAGCGCCCTACTCCACT (SEQ ID NO 4815) and DNA sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO 4816) are hybridised to form the dual siRNA structure ApoB-mTTR DNA sequences are indicated in bold GalNac = N-acetylgalactosamine fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mil, mC, mG = 2’ Methyl sugar base A, T, C, G = DNA bases rll, rG, rC, rA = RNA bases * internucleotide linkage (phosphorothioate (PS)). 24 Example 2 As shown in Table 4, DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers pre-incubated in vehicle showed high level of knockdown (KD) for both target genes DGAT2 and ApoB (KD range: 82.7% - 93%) when transfected in primary mouse hepatocytes at 5 concentration of 25 nM. When pre-incubated in 50% human serum followed by transfection in primary mouse hepatocytes at 25 nM, both DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers showed high KD levels fully comparable to pre-incubation in 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 target genes ApoB and 10 DGAT2 showed by DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers when preincubated in vehicle or 50% human serum followed by transfection, were fully comparable to the KD levels of their single siRNA control counterparts, ApoB_C3_OR, ApoB_REVCR, DGAT2_3 at 25 nM (KD range for siRNA controls: 90.2% - 93.7%). A full description of siRNA structures can be found in Table 5. 15 As for GS-mTTR-A and GS-mTTR-B heterodimers, pre-incubation in vehicle showed high levels of KD for both target genes ApoB and mTTR (KD range: 83.2% - 97%) when transfected in primary mouse hepatocytes at concentration of 25 nM. When preincubated in 50% human serum followed by transfection in primary mouse hepatocytes 20 at 25 nM, both GS-mTTR-A and GS-mTTR-B heterodimers showed high KD levels fully comparable to pre-incubation in vehicle (KD range after serum pre-incubation followed by transfection at 25 nM: 87.5% - 96.2%) suggesting high serum stability for these heterodimers. The KD levels of target genes ApoB and mTTR showed by GS-mTTR-A and GS-mTTR-B heterodimers when pre-incubated in vehicle or 50% human serum 25 followed by transfection, were fully comparable to the KD levels of their single siRNA control counterparts mTTR, mTTR-AC, mTTR-ARG, PAR-ApoB, GS-AC and GS-ARG (KD range for siRNA controls: 90.2% - 98.2%). A full description of siRNA structures used here can be found in Table 5. Table 4. Serum stability of heterodimer siRNAs DGAT2-ApoB and GS-mTTR and 30 standard siRNA controls siRNA ID KD levels (%) after pre-incubation in 50% human serum or vehicle 25 nM + vehicle 25 nM + 50% HS ApoB_C3_OR 91.4 92.1 ApoB_REVCR 91.6 90.2 DGAT2_3 93.7 91.8 DGAT2_9 95.3 17.3 DGAT2-ApoB-A (DGAT2) 93.0 86.9 DGAT2-ApoB-A (ApoB) 85.1 75.9 DGAT2-ApoB-B (DGAT2) 90.1 89.8 DGAT2-ApoB-B (ApoB) 82.7 79.4 mTTR 97.8 98.2 mTTR-AC 97.1 97.7 mTTR-ARG 96.8 97.4 PAR-ApoB 92.7 92.3 GS-AC 88.1 91.8 GS-ARG 90.5 90.2 GS-mTTR-A (ApoB) 86.7 89.5 GS-mTTR-A (mTTR) 95.2 96.2 GS-mTTR-B (ApoB) 86.1 87.5 GS-mTTR-B (mTTR) 96.2 96.1 24 Table 5. Description of control siRNAs and ApoB-mTTR heterodimer siRNAs siRNA ID Sense (5'-3') Antisense (5'-3') ApoB_C3_ OR CGAAGCGCCCTACTCCACT G*G*UGU AUG CU UCA ACC CUG A U*C*A GGG UUG AAG CCA UAC ACC*U*U ApoB_REV CR AGTGGAGTAGGGCGCTTCG G*G*UGU AUG GCU UCA ACC CUG A U*C*A GGG UUG AAG CCA rAC ACC*U*U DGAT2_3 CGAAGCGCCCTACTCCACTGUCA UGGGUGUCUGUGGGUUA UAACCCACAGACACCCAUGAC DGAT2_9 GUCAUGGGUGUCUGUGGGUUATC ACCTCATCCCGCGAAGC UAACCCACAGACACCCAUGAC DGAT2-ApoB_A heterodime r CGAAGCGCCCTACTCCACTG*U*C AUGGGUGUCUGUGGGUUA U*A*ACCCrCAGACACCCAUG*A* C AGTGGAGTAGGGCGCTTCGG*G*U GU AUG GCU UCA ACC CUG A U*C*A GGG UUG AAG CCA UAC ACC*U*U DGAT2- ApoB_B heterodime r AGTGGAGTAGGGCGCTTCGG*U*C AUGGGUGUCUGUGGGUUA U*A*ACCrACAGACACCCAUG*A* C CGAAGCGCCCTACTCCACTG*G*U GU AUG GCU UCA ACC CUG A U*C*A GGG UUG AAG CCA UAC ACC*U*U mTTR fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU mTTR_AC AGTGGAGTAGGGCGCTTCGfA*mA* fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU mTTR-ARG CGAAGCGCCCTACTCCACTfA*mA*f C mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU PAR_ApoB fC*mC*fU mGfGmA fCmAfU fUfCmA [VPm U*]fU* mCfUmU fGmUfU 24 fGmAfA mCfAmA fGmAfA -GalNAc mCfUmG mAmAfU mGfllmC fCmAfG mG*mG*mU GS_AC AGTGGAGTAGGGCGCTTCGfC*mC *fU mGfGmA fCmAfll fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU* mCfUmU fGmUfU mCfUmG mAmAfU mGfllmC fCmAfG mG*mG*mll GS_ARG CGAAGCGCCCTACTCCACTfC*mC* fU mGfGmA fCmAfU fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU* mCfUmU fGmUfU mCfUmG mAmAfU mGfUmC fCmAfG mG*mG*mU GS_ mTTR_A heterodime r CGAAGCGCCCTACTCCACTfC*mC* fU mGfGmA fCmAfU fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU*mCfUmU fGmUfU mCfUmG mAmAfU mGfUmC fCmAfG mG*mG*mU AGTGGAGTAGGGCGCTTCGfA*mA* fC mAfGmU fGmUfll fCfllmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU GS_ mTTR_B heterodime r AGTGGAGTAGGGCGCTTCGfC*mC *fU mGfGmA fCmAfU fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU*mCfUmUfGmUfUmCfUmGm AmAfUmGfUmCfCmAfGmG *mG*mU CGAAGCGCCCTACTCCACTfA*mA*f C mAfGmU fGmUfll fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU Notes: DNA sequence CGAAGCGCCCTACTCCACT and DNA sequence AGTGGAGTAGGGCGCTTCG are hybridised to form the heterodimer siRNAs DGAT2- ApoB and GS_ mTTR. fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mil, mC, mG = 2’ Methyl sugar base A, T, C, G = DNA bases rll, rG, rC, rA = RNA bases [VPmU*] - 5'-vinylphosphonate * internucleotide linkage (phosphorothioate (PS)). Example 3 5 As shown in Table 6, ApoB-mTTR_AG1 and ApoB-mTTR_AG2 heterodimers showed ApoB KD levels of 51.9% and 57% respectively at 100 nM and KD levels of 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, KD levels were 10 68.6% and 54.5% respectively suggesting that the heterodimer worked almost as effectively as single siRNA control in silencing target gene ApoB. Overall, ApoB-mTTR heterodimer structures showed to be highly effective in silencing mTTR target in all the ApoB-mTTR heterodimers tested here showing KD levels of mTTR ranging from 88.4% to 97.7% at 25 nM and 100 nM treatment in free-uptake assay. mTTR KD values showed by treatment with heterodimers were similar to KD values obtained with single mTTR 5 siRNA control treatment (KD level at 25 nM and 100 nM: 98.1%). A full description of siRNA structures used can be found in Table 7. 24 Table 6. Efficacy of GalNAc-conjugated ApoB-mTTR heterodimer siRNAs or standard siRNA controls by free-uptake in primary mouse hepatocytes siRNA ID Knockdown levels (%) of target gene by free-uptake 4 nM 25 nM 100 nM mTTR 97.3 98.1 98.1 ApoB_C3_01 23.2 54.5 68.6 ApoB-mTTR_AG1 (mTTR) 96.2 97.4 97.5 ApoB-mTTR_AG1 (ApoB) 37.1 48.0 51.9 ApoB-mTTR_AG2 (mTTR) 73.1 88.7 94.3 ApoB-mTTR_AG2 (ApoB) 29.5 46.2 57.0 ApoB-mTTR_AG3 (mTTR) 94.3 97.2 97.7 ApoB-mTTR_AG3 (ApoB) 10.2 14.2 9.3 ApoB-mTTR_BG1 (mTTR) 95.2 96.4 97.3 ApoB-mTTR_BG1 (ApoB) 12.9 24.8 43.6 ApoB-mTTR_BG2 (mTTR) 78.1 88.4 94.4 ApoB-mTTR_BG2 (ApoB) 20.2 15.8 43.1 ApoB-mTTR_BG3 (mTTR) 94.5 97.2 97.1 ApoB-mTTR_BG3 (ApoB) 4.5 0.0 0.0 15 Table 7. Standard control siRNAs and GalNAc-conjugated ApoB-mTTR heterodimer siRNA structures siRNA ID Sense (5'-3') Antisense (5'- 3') ApoB-mTTR_AG1 heterodimer GGUGU AUG GCU UCA ACC CUG A - GalNAc UCA GGG UUG AG rCCA UAC ACCUU AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU 24 ApoB-mTTR_AG2 heterodimer CGAAGCGCCCTACTCCACTrG*rG*rUrGrU rArllrG rGrCrll rllrCrA rArCrC rCrllrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfll mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_AG3 heterodimer GGUGU AUG GCU UCA ACC CUG A UCA GGG UUG AAG CCA UAC ACCUU AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_BG1 heterodimer AGTGGAGTAGGGCGCTTCGrG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU CGAAGCGCCCTACTCCACTfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_BG2 heterodimer AGTGGAGTAGGGCGCTTCGrG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU CGAAGCGCCCTACTCCACTfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_BG3 heterodimer GGUGU AUG GCU UCA ACC CUG A UCA GGG UUG AAG CCA UAC ACCUU CGAAGCGCCCTACTCCACTfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB_C3_01 GGUGU AUG GCU UCA ACC CUG A - GalNAc UCA GrG UUG AAG CCA UAC ACCUU mTTR fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA - GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU Notes: DNA sequence CGAAGCGCCCTACTCCACT and DNA sequence AGTGGAGTAGGGCGCTTCG in the heterodimer structures are hybridised to form the dual siRNA structure ApoB-mTTR fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mU, mC, mG = 2’ Methyl sugar base A, T, C, G = DNA bases rU, rG, rC, rA = RNA baseS Example 4 As shown in Table 8, ApoB-DGAT2 (CS15 and CS16) heterodimer siRNAs showed 5 ApoB KD levels of 64% and 60% respectively at 25 nM and KD levels of 49% and 50% respectively at 4 nM by receptor-mediated uptake in primary mouse hepatocytes. When cells were treated with single siRNA counterpart, CS13, ApoB KD was 72% and 53% respectively at 25nM and 4nM. For DGAT2 target KD, heterodimer siRNAs showed KD levels of 76% and 65% at 25nM and 55% and 57% at 4nM. In comparison, single siRNA CS8 led to 76% DGAT2 KD at 25nM and 48% at 4nM. 5 These results demonstrate that ApoB-DGAT2 heterodimer siRNAs perform as well as single ApoB or DGAT2 siRNAs of the same sequence, in receptor-mediated assays, leading to highly effective silencing of two targets in the same cell. A full description of siRNA structures used can be found in Table 9. Table 8 Target mRNA Knock-down (%) in primary mouse hepatocytes by 10 heterodimer siRNAs or single siRNA controls, with or without preincubation in 80 % human serum for 2 h at 37°C. siRNA ID Target gene mRNA KD levels (%) Freshly prepared Pre-incubated in 80% HS 25n M 4n M 1n M 0.1 n M 25n M 4n M 1n M 0.1n M CS1 3 PAR_ApoB_Cr ApoB 72 53 37 9 71 60 38 6 CS8 DGAT2_Acr DGAT 2 76 48 25 0 80 67 47 17 CS1 5 PAR_ApoB_Cr -DGAT2_Acr ApoB 64 49 37 10 71 52 31 6 DGAT 2 76 55 43 6 75 64 48 22 CS1 6 PAR_ApoB_Ac r-DGAT2_Cr ApoB 60 50 26 3 69 49 31 18 DGAT 2 65 57 32 1 73 58 39 0 PAR ApoB ApoB 69 61 45 20 66 64 49 2 mTTR mTTR 93 91 89 74 94 95 95 77 15 Example 5 20 To test stability in human serum, siRNA constructs were preincubated in 80% human serum (HS) for 2 hours at 37C and receptor-mediated uptake assays subsequently performed in primary mouse hepatocytes. Knockdown of target genes was measured after 48hrs by RT-qPCR. 25 As shown in Table 8, ApoB-DGAT2 heterodimer siRNAs, CS15 and CS16, showed equivalent levels of target KD when compared to single siRNAs of the same sequence, CS13 and CS8. ApoB KD was 71% and 73% respectively for CS15 and CS16 heterodimer siRNAs at 25nm and 52% and 49% at 4nM. This level of silencing compares favourably with that shown by single siRNA counterpart CS13; 71% and 60% KD at 25nM and 4nM, respectively. Similarly, levels of DGAT2 KD achieved by heterodimer 5 siRNAs was 75% and 73% at 25nM (64% and 58% at 4nM) versus 80% and 67% from single siRNA CS8 at 25nM and 4nM, respectively. These results demonstrate that ApoB-DGAT2 heterodimer siRNAs are highly stable in 80% human serum and perform as well as single siRNAs of the same sequence in 10 receptor-mediated uptake assays. A full description of siRNA structures used can be found in Table 9 below. 15 24 Table 9. ApoB-DGAT2 Heterodimer siRNAs and single siRNA constructs siR NA ID Sense (5’ - 3’) Antisense (5’ - 3’) CS13 PAR_ApoB_ Cr CGAAGCGCCCTACTCCAC 7TC*mC*fUmGfGmAfCmAfUf U fC m AfG m AfAm CfA m AfG m AfA-GalNAc mU*mG*mGfGmAfCmCfU mGfUmAmAmGfUmCfUm UfGmUfUmC*fU[VP mU] CS8 DGAT2_Acr AGTGGAGTAGGGCGCTTC GfG*mU*fCmAfUmGfGmGfU mGfUmCfUmGfUmGfGmGfU *mU*fA-GalNAc mC*fA*mGfUmAfCmCfCm AfCmAfGmAfCmAfCmCfC mA*fA*mU CS15 PAR_ApoB_ Cr-DGAT2_Acr CGAAGCGCCCTACTCCAC 7TC*mC*fUmGfGmAfCmAfUf UfCmAfGmAfAmCfAmAfGm AfA-GalNAc 1 [VPmU*]fU*mCfUmUfGmUf UmCfUmGmAmAfUmGfU mCfCmAfGmG*mG*mU AGTGGAGTAGGGCGCTTC GfG*mU*fCmAfUmGfGmGfU mGfUmCfUmGfUmGfGmGfU *mU*fA-GalNAc 1 mU*fA*mAfCmCfCmAfCm AfGmAfCmAfCmCfCmAfU mG*fA*mC CS16 PAR_ApoB_ Acr-DGAT2_Cr AGTGGAGTAGGGCGCTTC GfC*mC*fUmGfGmAfCmAfUf UfCmAfGmAfAmCfAmAfGm AfA-GalNAc 1 [VPmU*]fU*mCfUmUfGmUf UmCfUmGmAmAfUmGfU mCfCmAfGmG*mG*mU CGAAGCGCCCTACTCCAC 7TG*mU*fCmAfUmGfGmGfU mGfUmCfUmGfUmGfGmGfU *mU*fA-GalNAc 1 mU*fA*mAfCmCfCmAfCm AfGmAfCmAfCmCfCmAfU mG*fA*mC PAR_ApoB fC*mC*fUmGfGmAfCmAfUfU fCmAfGmAfAmCfAmAfGmAf A GalNAc mU*mG*mGfGmAfCmCfU mGfUmAmAmGfUmCfUm UfGmUfUmC*fU*[VPmu] mTTR fA*mA*fCmAfGmUfGmUfUfC fUmUfGmCfUmCfUmAfUmAf A-GalNAc mU*mU*mUfUmGfUmCfA mCfAmAmGmAfAmCfGmA fGmAfUmA*fU*mU 1 AGTGGAGTAGGGCGCTTCG (19 nt DNA fragment hybridised with complementary DNA at the 5' end of ApoB and DGAT2) * internucleotide linkage phosphorothioate (PS) [VPmll*] - 5'-vinylphosphonate GalNAc - N-acetylgalactosamine fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mil, mC, mG = 2’ Methyl sugar base A, T, C, G= DNA bases U, G, C, A = RNA bases 24 Example 4 Comparing in vivo silencing effect of heterodimer siRNA (ApoB-DGAT2) to single 5 siRNA controls. Mouse study Groups of 4 mice for each treatment group were injected subcutaneously (SC) with either vehicle (PBS), or GalNAc-conjugated ApoB-DGAT2 (CS15 or CS16) heterodimer, or ApoB (CS13) or DAGT2 (CS8) single siRNA controls (Table 2). Positive siRNA controls 10 included PAR-ApoB and mTTR (Table 2). Each compound was administered at either 5mg / kg or 10mg / kg (single siRNAs); equivalent to 10mg / kg and 20mg / kg, respectively, for heterodimer siRNAs. Following sacrifice at either day 5 or day 14, levels of liver target mRNA was measured by RT-15 qPCR and % knockdown (KD) measured relative to vehicle-treated controls. Table 10 shows in vivo silencing of liver target mRNAs (ApoB and DGAT2) by heterodimer siRNA compounds (CS15 and CS16) compared to single ApoB or DGAT2 siRNA controls (CS13 and CS8, respectively). 20 CS15 heterodimer (20mg / kg) led to a 53% KD of both ApoB and DGAT2 mRNA at Day 5, with CS16 heterodimer providing 62% and 54% KD of ApoB and DGAT2, respectively. Single siRNAs, CS13 and CS8, at the equivalent dose (10mg / kg) led to 61% and 49% KD of ApoB and DGAT2, respectively. A positive control siRNA (PAR-ApoB; Alnylam) 25 performed similarly, leading to 63% ApoB KD at Day 5. The silencing of ApoB was reduced at Day 14 for heterodimer CS15 and single siRNA CS13 (13% and 11%, respectively) which was not unexpected, given the positive control of the same sequence (PAR-ApoB; Alnylam) performed similarly (17% KD). The heterodimer CS16 however, provided a KD of 34% at D14. In contrast, silencing of DGAT2 mRNA was maintained in mice receiving either heterodimer (CS15 67% KD; CS16 57%) or single siRNA CS8 (51%) at Day14. Importantly, there was a significant increase in silencing of DGAT2 by CS15 heterodimer (Day 14) at both doses when compared to single DGAT2 siRNA CS8 (P=0.02 lower 5 dose; P=0.008 higher dose). For CS16 heterodimer at the lower dose, ApoB knockdown at Day14 was significantly greater when compared to single ApoB siRNA CS13 (P= 0.04). Statistical analysis performed: 10 Two-way Anova and Tukey post-hoc tests These results demonstrate that both ApoB and DGAT2 target KD (%) in vivo by heterodimer siRNAs is equivalent or superior to single siRNA controls at both low (5mg / kg) and high dose (10mg / kg), and at both time points (day 5 and day 14). 15 20 24 Table 10. Mouse study comparing knock-down (%KD) of liver target mRNA following SC administration of either ApoB-DGAT2 heterodimer siRNA or equivalent single ApoB or DGAT2 siRNAs siRNA ID Primer used Dose (mg / kg) % KD On day 5 % KD On day 14 CS13 PAR_ApoB_Cr ApoB 5 26 7 10 61 11 CS8 DGAT2_Acr DGAT2 5 33 31 10 49 51 CS15 PAR_ApoB_Cr-DGAT2_Acr ApoB 10 34 15 DGAT2 10 41 46 ApoB 20 53 13 DGAT2 20 53 67 CS16 PAR_ApoB_Acr-DGAT2_Cr ApoB 10 42 20 DGAT2 10 28 32 ApoB 20 62 34 DGAT2 20 54 57 PAR_ApoB ApoB 5 44 0 10 63 17 mTTR mTTR 10 91 70 Table 11 shows Comparative Efficacy (%) of ApoB-DGAT2 heterodimer siRNA 25 compared to each single siRNA (ApoB or DGAT2) on day 5 and day 14. [%KD ApoB or DGAT2 by heterodimer siRNA divided by %KD by mono siRNA (x100)]. Equivalent or superior performance is achieved by heterodimer siRNAs compared to single siRNA controls. At the lower dose, for ApoB KD, heterodimers shows efficacy of 134 - 164% (Day 5) and 210 - 280% (Day 14) and at higher dose; 87-102% (Day 5) and 114 - 298% (D14). For DGAT2 KD, at the lower dose, comparative efficacy is 84 - 124% 5 (Day 5) and 102 - 147% (Day 14); whereas at higher dose, 109 - 111% (Day 5) and 112 -131% (Day 14). Table 11 Comparative efficacy (%) of ApoB-DGAT2 heterodimer siRNAs to single ApoB or DGAT2 siRNAs Bispecific Target mRNA Low dose High dose Day 5 Day 14 Day 5 Day 14 CS15 ApoB 134 210 87 114 CS16 ApoB 164 280 102 298 CS15 DGAT2 124 147 109 131 CS16 DGAT2 84 102 111 112 15 Example 5: alternative sequences 24 Table 12 Sequence Sense (5’-3’) Antisense (5’-3’) Inclisiran mC *mU *mAmGmAmCfCmUfGmUdTmUmUmGmCm UmUmUmUmGmU (SEQ ID NO 4821) mA* fC *mAfAfAfArnGfCmAfArnAf AmC f AmG f GmU f CmUmAmG * mA * mA (SEQ ID NO 4822) SLN360 mCmGmGmUmAmAfUfG fGmAmCmAmGmAmGmUm U*mA*mU (SEQ ID NO 4823) mA*fU*mAfAmCfUmCfUmGfUmfC mAfUmUfAmC*fC*mG (SEQ ID NO 4824) ARO-ANG3 (invAb)*mGmCmUmCmAmAmCmAfUfAfUmUm UmGmAmUmCmAmGmUmA*(invAb) (SEQ ID NO 4825) mU*fA*mC*fUmGfAmUfCmAfAmA fUmAfUmGfUmUfGmAfG*mC (SEQ ID NO 4826) ANGsiRIO CCAAGAGCACCAAGAACUA(SEQ ID NO 4827) UAGUUCUUGGUGCUCUUGGCU(SEQ ID NO 4828) DGAT2-1473 mU * mG * mGmGmUmU mA fU fU fUmA fAmAmAmGm A *mA*mA (SEQ ID NO 4829) mU* fU *mUmCmUfUmUmUmAmAmAm UmA*fA*mC*fC*mC*mA*mC*fA (SEQ ID NO 4830) fA, fll, fC, fG = 2’-F ribonucleotides 20 mA, mU, mC, mG = 2’ OMe ribonucleotides dT = Thymidine U = Vinyl phosphonate (invAb) = inverted abasic deoxyribose Phosphorothioate 24 Table 13 5 Sequence Sense (5’-3’) Antisense (5’-3’) Inclisiran PCSK9 Alnylam mC *mU *mAmGmAmCfCmU fGmUdTm UmUmGmCmUmUmUmUmGmU (S EQ ID NO 4821) mA*fC*mAfAfAfAmGfCmAfAm AfAmCfAmGf GmUfCmUmAmG *m A*mA(SEQ ID NO 4822) Crook-lnclisiran CGAAGCGCCCTACTCCACTmC*mU* mAmGmAmC f CmUfGmUdTmUmUmGm CmUmUmUmUmGmU (SEQ ID NO 4849) mA* fC *mAfAfAfAmG fCmAfAm AfAmCfAmGf GmUfCmUmAmG*m A*mA(SEQ ID NO 4822) Anticrook-lnclisiran AGTGGAGTAGGGCGCTTCGmC*mU* mAmGmAmC f CmU fGmUdTmUmUmGm CmUmUmUmUmGmU(SEQ ID NO 4850) mA*fC*mAfAfAfAmGfCmAfAm AfAmCfAmGfGmUfCmUmAmG*m (SEQ ID NO 4822) SLN360 Lp(a) Silence mCmGmGmUmAmAfUfG fGmAmCmAm GmAmGmUmU*mA*mU (SEQ ID NO 4823) mA*fU*mAfAmCfUmCfUmGfUm fC mAfUmUfAmC*fC*mG (SEQ ID NO 4824) Crook-SLN360 CGAAGCGCCCTACTCCACTmCmGmG mUmAmAfUf GfGirAmCmAmGmAmGm UmU*mA*mU(SEQ ID NO 4851) mA*fU*mAfAmCfUmCfUmGfUm fC mAfUmUfAmC*fC*mG (SEQ ID NO 4824) Anticrook-SLN360 AGT GGAGTAGGGCGCT T C GmCmGmG mUmAmAfU f G fGmAmCmAmGmAmGm UmU*mA*mU (SEQ ID NO 4852) mA*fU*mAfAmCfUmCfUmGfUm fC mAfUmUfAmC*fC*mG (SEQ ID NO 4824) AR0-ANG3 ANGPTL3: Arrowhead; Zodasiran *(invAb)*mGmCmUmCmAmAmCmA fUfAfUmUmUmGmAmUmCmAmGmUm A*(invAb) (SEQ ID NO 4825) mU*fA*mC*fUmGfAmUfCmAfA mAfUmAfUmGfUmUfGmAfG*mC (SEQ ID NO 4826) Crook-ARO-ANG3 CGAAGCGCCCTACTCCACT*(invA b)*mGmCmUmCmAmAmCmAfUfAfU mUmUmGmAmUmCmAmGmUmA*(inv Ab) (SEQ ID NO 4853) mU*fA*mC*fUmGfAmUfCmAfA mAfUmAfUmGfUmUfGmAfG*mC (SEQ ID NO 4826) Anticrook-ARO- ANG3 AGTGGAGTAGGGCGCTTCG*(invA b)*mGmCmUmCmAmAmCmAfUfAfU mUmUmGmAmUmCmAmGmUmA*(inv Ab) (SEQ ID NO 4854) mU* fA*mC * fUmGfAmUfCmAfA mAfUmAfUmG fUmU f GmAf G*mC (SEQ ID NO 4826) ANGsiRIO ANGPTL3 Suzhou Ribo CCAAGAGCACCAAGAACUA ( SEQ ID NO 4827) UAGUUCUUGGUGCUCUUGGCU ( SEQ ID NO 4828) Crook-ANGsiR10 CGAAGCGCCCTACTCCACTCCAAGA UAGUUCUUGGUGCUCUUGGCU( GCACCAAGAACUA ( SEQ ID NO 4855) SEQ ID NO 4828) Anticrook- ANGsiRIO AGT GGAGTAGGGCGCT T C GC CAAGA GCACCAAGAACUA( SEQ ID NO 4856) UAGUUCUUGGUGCUCUUGGCU ( SEQ ID NO 4828) DGAT2-1473 Khvorova, A mU*mG*mGmGmUmUmAfUfUfUmAf AmAmAmGmA*mA*mA (SEQ ID NO 4829) mt- * f u *mUmCmU fUmUmUmAmAm AmUmA*fA*mC*fC*mC*mA*mC *fA (SEQ ID NO 4830) Crook-DGAT2-1473 CGAAGCGCCCTACTCCACTmU*mG* mGmGmUmUmAfU fU fUmAfAmAmAm GmA*mA*mA (SEQ ID NO 4857) mU* fU*mUmCmU fUmUmUmAmAm AmUmA*fA*mC*fC*mC*mA*mC *fA (SEQ ID NO 4830) Anticrook-DGAT2-1473 AGTGGAGTAGGGCGCTTCGmU*mG* mGmGmUmUmAfU fU fUmAfAmAmAm GmA*mA*mA (SEQ ID NO 4858) mU* fU*mUmCmU f UmUmUmAmAm AmUmA*fA*mC*fC*mC*mA*mC *fA (SEQ ID NO 4830) fA, fll, fC, fG = 2’-F ribonucleotides mA, mil, mC, mG = 2’ OMe ribonucleotides dT = Thymidine U = Vinyl phosphonate 5 (invAb) = inverted abasic deoxyribose *Phosphorothioate 24 Table 14: DGAT 2 Lead Sequences Sense Antisense AAGAAGUUCCAGAAAUACAA (SEQ ID NO 4831) UUGGAGAGAAUGAAGUGUAA(SEQ ID NO 4832) CUCAUGUACAUAUUCUGCAA (SEQ ID NO 4833) ACCAUAGACUAUUUGCUUUA(SEQ ID NO 4834) CCAAGAAGUUCCAGAAAUACAA(SEQ ID NO 4835) CCCAUAGACUAUUUGCUUUCAA(SEQ ID NO 4836) CCUUGGAGAGAAUGAAGUGUA(SEQ ID NO 4837) CCCUCAUGUACAUAUUCUGCAA(SEQ ID NO 4838) CCACCAUAGACUAUUUGCUUUA (SEQ ID NO 4839) UUGUAUUUCUGGAACUUCUU(SEQ ID NO 4840) UUACACUUCAUUCUCUCCAA(SEQ ID NO 4841) UUGCAGAAUAUGUACAUGAG(SEQ ID NO 4842) UAAAGCAAAUAGUCUAUGGU(SEQ ID NO 4843) UUGUAUUUCUGGAACUUCUUGG(SEQ ID NO 4844) UUGAAAGCAAAUAGUCUAUGGG(SEQ ID NO 4845) UACACUUCAUUCUCUCCAAGG(SEQ ID NO 4846) UUGCAGAAUAUGUACAUGAGGG(SEQ ID NO 4847) UAAAGCAAAUAGUCUAUGGUGG(SEQ ID NO 4848) Table 15 5 ApoC3 antisense strand: usCfsasCfuGfagaauAfcUfgUfcCfcGfsu (SEQ ID NO:4859) sense strand: (invAb)sacgggacaGfUfAfuucucaguias(invAb) (SEQ ID NO:4860) 10 Crook: CGAAGCGCCCTACTCCACT (invAb)sacgggacaGfllfAfuucucaguias(invAb) (SEQ ID NO:4861) Anticrook: 15 AGTGGAGTAGGGCGCTTCG (invAb)sacgggacaGfUfAfuucucaguias(invAb) (SEQ ID NO:4862) a, c, g, i, and u represent 2'-O-methyl adenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, cytidine, 20 guanosine, and uridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted abasic deoxyribose residue. 25 30 35 40 45 50 24

Claims

1. A nucleic acid molecule comprising:i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a cardiovascular disease associated gene to be silenced wherein said cardiovascular gene is diacylglycerol O acyltransferase 2 (DGAT 2) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand; andii) a second nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a different cardiovascular disease associated gene to be silenced wherein said different cardiovascular gene is angiopoietin like 3 (ANGPTL 3) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals 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. The nucleic acid molecule according to claim 1 wherein said DGAT2 gene comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

3. The nucleic acid molecule according to claim 2 wherein said double stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 7 to 306.

4. The nucleic acid molecule according to claim 2 wherein said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 307 to 606.

5. The nucleic acid molecule according to claim 2 wherein said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4830.

246. The nucleic acid molecule according to claim 2 wherein said double stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence set forth in SEQ ID NO: 4829.

7. The nucleic acid molecule according to claim 2 wherein said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4840 to 4848.

8. The nucleic acid molecule according to claim 2 wherein said double stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 4831 to 4839.

9. The nucleic acid molecule according to any one of claims 1 to 8 wherein said ANGPTL3 gene comprises a nucleotide sequence set forth in SEQ ID NO: 4819 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

10. The nucleic acid molecule according to claim 9 wherein said double stranded ANGPTL 3 inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 3615 to 3914.

11. The nucleic acid molecule according to claim 9 wherein said double stranded ANGPTL 3 inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 3915 to 4214.

12. The nucleic acid molecule according to claim 9 wherein said double stranded ANGPTL3 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4826 or 4828.

13. The nucleic acid molecule according to claim 9 wherein said double stranded ANGPTL3 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 4825 or 4827.

14. The nucleic acid molecule according to any one of claims 1 to 13 wherein said first and / or said second double stranded inhibitory (RNA) molecule comprises modified nucleotides and / or modified sugar(s).2415. The nucleic acid molecule according to claim 1 wherein said nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830).

16. The nucleic acid molecule according to claim 1 wherein said nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826).

17. The nucleic acid molecule according to claim 1 wherein said nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4827) and an antisense strand (SEQ ID NO: 4828).

18. The nucleic acid molecule according to claim 1 wherein said nucleic acid molecule comprises:i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826); andii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830).

19. The nucleic acid molecule according to any one of claims 1 to 14 wherein said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815).

20. The nucleic acid molecule according to any one of claims 1 to 14 wherein said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816).

21. The nucleic acid molecule according to claim 1 wherein said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4853 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4826.2422. The nucleic acid molecule according to claim 1 to wherein said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4854 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4826.

23. The nucleic acid molecule according to claim 1 wherein said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4855 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4828.

24. The nucleic acid molecule according to claim 1 wherein said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4856 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4828.

25. The nucleic acid molecule according to claim 1 wherein said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4857 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4830.

26. The nucleic acid molecule according to claim 1 wherein said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4858 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4830.27 The nucleic acid molecule according to any one of claims 1 to 26 wherein said nucleic acid molecule is covalently linked to N-acetylgalactosamine.

28. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 27 and including a pharmaceutical carrier and / or excipient.

29. A nucleic acid molecule or a pharmaceutical composition according to any one of claims 1 to 28 for use in the treatment or prevention of a subject that has or is predisposed to hypercholesterolemia.

30. The nucleic acid molecule or pharmaceutical composition for use according to claim 29 in the treatment or prevention of diseases associated with hypercholesterolemia selected from the group consisting of: stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, aortic stenosis, cerebrovascular disease, peripheral arterial disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Buerger’s disease, renal artery stenosis, hyper-apobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis.24